Topical compositions of Micrococcus luteus Q24 and their uses

Abstract

The present invention relates to topical compositions comprising Micrococcus luteus Q24 and a viscosity adjusting agent, the topical compositions having a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C, and uses thereof for improving the appearance of skin or at least one sign of aging or reducing dryness of the skin.

Description

[Technical Field]

[0001] This application claims priority to Australian Provisional Patent Application No. 2022902530 filed on 2 September 2022, the contents of which are incorporated herein by reference.

[0002] The present invention relates to Micrococcus luteus compositions and methods of using the compositions to improve the appearance of skin or at least one sign of skin aging. The present invention also relates to methods of making the compositions. [Background technology]

[0003] Skin care products containing probiotic microorganisms are becoming increasingly known. To date, the microorganisms or related products used in skin care products are generally Bifidobacterium species, Lactobacillus species (also known as Limosilactobacillus species, Lacticaseibacillus species, Lactiplantibacillus species, and Ligilactobacillus species), Lactococcus species, and Streptococcus species, or filtrates or lysates produced from these bacteria. However, the role they play in interacting with skin microflora is not fully understood.

[0004] WO 2006104403 (Blis Technologies Limited) describes Micrococcus luteus (M. luteus) compositions and their therapeutic use for controlling skin diseases or disorders. Also provided is the probiotic strain Q24, deposited with Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, under accession number DSM 17172. This document is incorporated herein by reference in its entirety. WO 2006104403 discloses a saline suspension, a grape seed oil suspension, and a hard deodorant stick.

[0005] The ANZCTR's A Probiotic for Eczema Treatment (registration number: ACTRN12616000022460) describes a clinical trial testing the use of Micrococcus luteus Q24 lysate for the treatment of eczema. No results are provided.

[0006] Applicants have unexpectedly identified a novel role for Micrococcus 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.

[0007] Working with probiotic organisms can be challenging: probiotics are sensitive and often react unpredictably to various freeze-drying processes, the chemicals used in such processes, processing conditions (such as pressure or high temperature), and formulation agents.

[0008] During the harsh freeze-drying process, microorganisms are typically protected using various lyoprotectants and cryoprotectants. Applicants have also surprisingly found that Micrococcus luteus can be formulated in the absence of lyoprotectants or cryoprotectants without significantly compromising the viability of the probiotic microorganism.

[0009] Preparing compositions containing Micrococcus luteus can be particularly challenging because Micrococcus luteus has been found to be unstable in aqueous and polar solvents and carriers and is sensitive to chemicals and heat. One solution to the viability problem is two-part formulations, in which the live microbial component is mixed with the solvent or carrier component immediately before use. Disadvantages of this approach include increased cost and complexity of the manufacturing process, reduced user convenience, and the need for additional packaging. Therefore, there remains a need for cosmetic compositions that contain all components, including live, viable Micrococcus luteus Q24, in a single formulation.

[0010] A particular challenge in working with Micrococcus luteus is the preparation of compositions with highly viscous components that require heating or melting during preparation, which can kill or adversely affect the viability of Micrococcus luteus.

[0011] Another issue is the requirement of a suitable formulation that maintains the microbiological stability and viability of Micrococcus luteus and provides good temporal, thermal and physical stability of the formulation.

[0012] Consumer demand for preservative-free and / or anti-allergenic products presents additional challenges by limiting the choice of formulation ingredients and increasing the difficulty of providing the desired properties in the resulting formulation. Summary of the Invention [Problem to be solved by the invention]

[0013] It is an object of the present invention to provide a Micrococcus luteus composition that goes at least some way toward addressing one or more of the above problems and / or at least provides the public with a useful choice.

[0014] Other objects of the present invention will become apparent from the following description, which is given by way of example only.

[0015] Any discussion of documents, acts, materials, devices, articles or the like which has been 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 such matters by virtue of existing prior to the priority date form part of the body of prior art or were common general knowledge in the art relevant to the present invention. [Means for solving the problem]

[0016] In a first aspect, the present invention provides a topical composition comprising Micrococcus luteus Q24 and a viscosity adjusting agent, the topical composition having a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C.

[0017] In a second aspect, the present invention provides a topical composition comprising: Approximately 0.5-2% w / w of Micrococcus luteus Q24, about 15 to about 35% w / w beeswax, about 2 to about 10% w / w cetostearyl alcohol, about 10% to about 30% w / w cocoa butter, A sufficient amount of oily base, Including, The composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C. A topical composition is provided.

[0018] In a third aspect, the present invention provides a topical composition comprising: Approximately 1×10 3 ~Approx. 1×10 12 cfu / g Micrococcus luteus Q24, about 15 to about 35% w / w beeswax, about 2 to about 10% w / w cetostearyl alcohol, about 10% to about 30% w / w cocoa butter, A sufficient amount of oily base, Including, The composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C. A topical composition is provided.

[0019] In a fourth aspect, the present invention provides a topical composition comprising: Approximately 0.5-2% w / w of Micrococcus luteus Q24, about 10 to about 30% w / w beeswax, about 10% to about 30% w / w shea butter, about 10% to about 30% w / w cocoa butter, A sufficient amount of oily base, Including, The composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C. A topical composition is provided.

[0020] In a fifth aspect, the present invention provides a topical composition comprising: Approximately 1×10 3 ~Approx. 1×10 12 cfu / g Micrococcus luteus Q24, about 10 to about 30% w / w beeswax, about 10% to about 30% w / w shea butter, about 10% to about 30% w / w cocoa butter, A sufficient amount of oily base, Including, The composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C. A topical composition is provided.

[0021] In a sixth aspect, the present invention provides a method of improving the appearance of skin or at least one sign of aging, comprising applying to the skin a topical composition according to the first, second or third aspect.

[0022] In a seventh aspect, the present invention provides a topical composition according to the first, second or third aspect for improving the appearance of skin or at least one sign of aging, or alleviating dryness of the skin.

[0023] In an eighth aspect, the present invention provides the use of a topical composition according to the first, second or third aspect in the manufacture of a medicament for improving the appearance of skin or at least one sign of aging, or reducing dryness of the skin.

[0024] In a ninth aspect, the present invention provides a method for preparing a topical composition comprising Micrococcus luteus Q24, the method comprising: a) heating a mixture of an oily base and a viscosity modifier to provide a molten mixture; b) cooling the molten mixture to a temperature in the range of about 35°C to about 28°C; c) adding Micrococcus luteus Q24 to the molten mixture from step b) to provide a homogenous mixture; d) cooling the mixture from step c) to room temperature to provide the composition. The present invention provides a method of manufacturing a semiconductor device, comprising:

[0025] In a tenth aspect, the present invention provides a method for preparing a topical composition comprising Micrococcus luteus Q24, the method comprising: a) heating a mixture of an oily base and a viscosity modifier to provide a molten mixture; b) cooling the molten mixture to a temperature of about 22°C to about 27°C; c) adding Micrococcus luteus Q24 to the cooled mixture from step b) and mixing to provide a homogenous mixture. The present invention provides a method of manufacturing a semiconductor device, comprising:

[0026] The following embodiments and preferences may relate to any of the above aspects, either alone or in any combination of any two or more.

[0027] In various embodiments, the composition comprises about 1 x 10 Micrococcus luteus Q24. 3 ~Approx. 1×10 12 Contains cfu / g.

[0028] In various embodiments, the composition comprises one or more viscosity modifiers in a total amount of about 30 to about 80% w / w.

[0029] In various embodiments, the viscosity modifier is selected from the group consisting of hydrophobic silica, hydrophilic silica, white wax (cera alba), yellow wax (cera alba), ethyl cellulose, emulsifying wax, cocoa butter, shea butter, fatty alcohols, glyceryl monostearate, candelilla wax, lanolin, and combinations of any two or more thereof. In addition to being a viscosity modifier, lanolin functions as a skin protectant.

[0030] In various embodiments, the viscosity modifier is selected from a combination of white wax or yellow wax and cetostearyl alcohol.

[0031] In various embodiments, the composition includes a dispersant.

[0032] In various embodiments, the composition comprises a dispersant in an amount of about 0.1 to about 5% w / w.

[0033] 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, sodium stearoyl glutamate, sodium cocoyl isethionate, cetearyl olivate, and combinations of any two or more thereof.

[0034] In various embodiments, the composition comprises about 0.5 to about 2% w / w polysorbate 80.

[0035] In various embodiments, the composition further comprises an oily base.

[0036] In various embodiments, the oily base is selected from the group consisting of medium chain triglyceride (MCT) oil, vegetable oil, or a combination thereof.

[0037] In various embodiments, the MCT oil is caprylic / capric triglyceride oil.

[0038] In various embodiments, the vegetable oil is selected from the group consisting of sunflower oil, canola oil, soybean oil, olive oil, jojoba oil, argan oil, rosehip oil, marula oil, chamomile oil, tamanu oil, grapeseed oil, calendula oil, pomegranate oil, macadamia oil, barberry fruit oil, sweet almond oil, evening primrose oil, and combinations of any two or more thereof.

[0039] In various embodiments, the vegetable oil is selected from the group consisting of MCT oil, olive oil, calendula oil, pomegranate oil, and combinations of any two or more thereof.

[0040] In various embodiments, the vegetable oil is MCT oil or olive oil.

[0041] In various embodiments, the Micrococcus luteus Q24 does not contain a lyoprotectant.

[0042] In various embodiments, the composition further comprises one or more additional probiotic(s).

[0043] In various embodiments, the additional probiotic(s) is selected from the group consisting of Streptococcus spp., Lactobacillus spp., Rimosilactobacillus spp., Lacticaseibacillus spp., Ligilactobacillus spp., Lactipranchibacillus spp., Bifidobacterium spp., Saccharomyces spp., and combinations of any two or more thereof.

[0044] In various embodiments, the Streptococcus species is selected from the group consisting of Streptococcus salivarius K12, Streptococcus salivarius M18, Streptococcus salivarius 24 SMB, Streptococcus oralis (e.g., S. oralis 89a), Streptococcus salivarius DB-B5, and combinations of any two or more thereof.

[0045] In various embodiments, the composition contains about 1 x 10 each additional probiotic. 3 ~Approx. 1×10 12 Contains cfu / g.

[0046] In various embodiments, the composition further comprises an inhibitory activity enhancer, a buffering agent, an antimicrobial agent, a prebiotic, a fragrance, an antioxidant, a colorant, a skin protectant, an antimicrobial agent, an aluminum salt, an inorganic pigment, an odor absorber or neutralizer, a sunscreen, and combinations of any two or more thereof.

[0047] 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.

[0048] 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 oxides, bentonite clay, kaolin clay, and combinations thereof.

[0049] In various embodiments, the antimicrobial 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 antimicrobial agent is xylitol, erythritol, epidermin, nisin, salivaricin A, salivaricin A1, salivaricin A2, salivaricin B, salivaricin 9, salivaricin MPS, salivaricin D (Birri et al., Appl Environ Microbiol. 2012 Jan;78(2):402-410; GenBank Accession No. JN564797), salivaricin M18, (Wescombe et al., Future Microbiol. 2012;7(12):1355-1371; American Type Culture Collection, Virginia, United States, Accession No. BAA 2593; Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, Accession No. DSM 14685), salivaricin G32 (Wescombe et al., International Journal of Microbiology, vol. 2012, Article ID 738503, 2012; GenBank accession number JN831266), and combinations thereof.

[0050] In various embodiments, the prebiotic(s) is selected from the group consisting of manuka honey powder, olive squalene, 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, colloidal oatmeal, xylitol, yeast extract, fructooligosaccharide powder, fructooligosaccharide liquid, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, niacinamide, sunscreen, and combinations of any two or more thereof.

[0051] In various embodiments, the prebiotic(s) is selected from the group consisting of yeast extract, cysteine, maltodextrin, inulin, liquorice root, licorice extract, honey, and combinations of any two or more thereof.

[0052] In various embodiments, the composition comprises 0.1-35% w / w prebiotic(s).

[0053] In various embodiments, the prebiotic(s) is selected from olive squalene, pomegranate seed oil, vitamin E, or a combination thereof.

[0054] In various embodiments, the prebiotic(s) is selected from a combination of olive squalene and pomegranate seed oil; a combination of pomegranate seed oil and oatmeal; a combination of olive squalene and vitamin E; and a combination of olive squalene, pomegranate seed oil and vitamin E.

[0055] In various embodiments, the composition is preservative-free.

[0056] In various embodiments, the composition further comprises one or more additional postbiotic(s).

[0057] In various embodiments, the postbiotic is a fermentate, filtrate, supernatant, fermentation filtrate, or lysate.

[0058] In various embodiments, the postbiotic(s) is selected from the group consisting of Streptococcus ferment, Streptococcus fermentation filtrate, Streptococcus fermentation lysate, Streptococcus lysate, Streptococcus filtrate, Lactobacillus (including Rimosilactobacillus spp., Ligilactobacillus spp., and Lactiprancylbacillus spp.) ferment, Lactobacillus (including Rimosilactobacillus spp., Ligilactobacillus spp., and Lactiprancylbacillus spp.) filtrate, Micrococcus fermentation lysate, Bifidobacterium fermentation lysate, Bifidobacterium fermentation filtrate, Galactomyces fermentation filtrate, Saccharomyces fermentation filtrate, Bacillus ferment, Bacillus filtrate, and combinations of any two or more thereof.

[0059] 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.

[0060] 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, bakuchiol, licorice root, and combinations of any two or more thereof.

[0061] In various embodiments, the antioxidant is selected from olive squalene, 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.

[0062] 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, palmitic acid heptapeptide-15, 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, sunscreen, lactic acid, camellia oil, hemp seed oil, seabuckthorn fruit oil, lanolin, and combinations of any two or more thereof.

[0063] In various embodiments, the antimicrobial agent is selected from the group consisting of zinc, salicylic acid, azelaic acid, benzoyl peroxide, tea tree oil, salicylic acid, and combinations thereof.

[0064] In various embodiments, the composition further comprises about 0.1% to about 10% w / w olive squalene and about 0.1 to about 10% w / w pomegranate seed oil.

[0065] In various embodiments, the composition further comprises about 0.1% to about 10% w / w olive squalene and about 0.1 to about 3% w / w vitamin E.

[0066] In various embodiments, the composition comprises about 0.1% to about 10% w / w olive squalene, and about 0.1 to about 10% w / w pomegranate seed oil, and about 0.1 to about 3% w / w vitamin E.

[0067] In various embodiments, improving the appearance of 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 looking clearer, reduced wrinkles, less dryness, reduced blemishes, reduced impurities, increased moisture, reduced exfoliation, and regulated sebum production.

[0068] In various embodiments, improving the appearance of the skin includes improving at least one of teething rash, diaper rash, or dry skin in infants.

[0069] In various embodiments, improving the appearance of skin or at least one sign of aging comprises reducing dryness of the skin.

[0070] In various embodiments, the composition is non-aqueous.

[0071] In various embodiments, the composition has a shelf life of at least 3 months at 25° C. and 60% RH. In various embodiments, the composition has a shelf life of at least 12 months at 25° C. and 60% RH.

[0072] In various embodiments, the oily base is a medium chain triglyceride.

[0073] 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).

[0074] In various embodiments, the prebiotic(s) is selected from olive squalene, pomegranate seed oil, vitamin E, or a combination thereof.

[0075] In various embodiments, the composition further comprises 0.1-10% w / w of postbiotic(s). In various embodiments, the composition further comprises 0.1-35% w / w of postbiotic(s).

[0076] In various embodiments, the prebiotic is selected from a combination of olive squalene and pomegranate seed oil; a combination of olive squalene and vitamin E; and a combination of olive squalene, pomegranate seed oil, and vitamin E.

[0077] In various embodiments, the particle size (Dv90) of Micrococcus luteus Q24 is less than about 300 μm.

[0078] In various embodiments, the particle size of the Micrococcus luteus Q24 is less than about 250 μm or less than about 100 μm.

[0079] In various embodiments of the ninth aspect, the molten mixture is cooled to about 30° C. in step (b).

[0080] In various embodiments of the tenth aspect, the molten mixture is cooled to room temperature in step (b).

[0081] In various embodiments of the ninth and tenth aspects, the method further comprises adding a dispersant during step (a) or before step (b).

[0082] In various embodiments of the seventh and eighth aspects, the topical composition produced is a composition of the first, second, third, fourth, fifth or seventh aspect.

[0083] The present invention may also be broadly stated as consisting of any or all combinations of two or more of the parts, elements and features referred to or indicated in the specification of this application, individually or collectively, of such parts, elements or features, and where a specific integer having known equivalents in the art to which the invention pertains is referred to herein, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0084] Reference to a range of numbers disclosed herein (e.g., 1 to 10) also incorporates reference to every rational number within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10), and also to any range of rational numbers within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus all subranges of every range explicitly disclosed herein are intended to be hereby expressly disclosed. These are merely examples of what is specifically intended, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited should likewise be considered to be expressly stated in this application.

[0085] Where reference is made herein to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing features of the present invention. Unless otherwise expressly stated, the reference to such external documents should not be construed as an admission that such documents or such sources are prior art in any jurisdiction or form part of the common general knowledge in the art.

[0086] Numerous modifications of the structure and a wide variety of embodiments and applications of the present invention will suggest themselves to those skilled in the art to which this invention pertains without departing from the scope of the invention, which is defined in the appended claims. The disclosures and descriptions herein are purely illustrative and are not intended to be in any sense limiting.

[0087] Although the present invention is broadly defined as above, those skilled in the art will appreciate that the invention is not so limited, but also includes embodiments of which the following description provides examples. [Brief explanation of the drawings]

[0088] The present invention can be described with reference to the following accompanying figures. [Figure 1]Shelf life stability of Micrococcus luteus Q24 lyophilized raw material components under refrigerated storage conditions at 4°C. P1: Trehalose as lyoprotectant. P2: Blis Technologies Ltd's trimix blend of trehalose, maltodextrin and lactitol as lyoprotectants. P3: No lyoprotectant. [Figure 2] Stability data for balm formulations over a 3-month period at 25°C ± 2°C / 60% RH are shown. [Figure 3] Figure 1 shows the microbial cell counts of the deodorant stick disclosed in WO 2006 / 104403 under controlled storage conditions (25°C / 60%RH) over a period of 4 months. [Figure 4] The malleability (percentage (%) increase in diameter) of BLT-BB5, BLT-BB9 and BLT-BB2 is shown. [Figure 5A] Table 1 shows graphed cumulative syneresis at different centrifugation speeds (1000, 1500, 5000, and 13000 rpm) at 20 minutes for selected formulations shown in Table 1 and a deodorant stick prepared according to Example 2 of WO 2006 / 104403 (trace "Deodorant Stick") The cumulative syneresis includes the percent oil leakage released at all time points, including before and at 20 minutes. [Figure 5B] Table 4 shows graphed cumulative syneresis at different centrifugation speeds (1000, 1500, 5000 and 13000 rpm) at 20 minutes for serum formulations containing 4, 5, 6 and 7% hydrophobic silica. [Figure 6] A scatterplot showing tissue viability (compared to the PBS negative control treatment) for subjects receiving each dose level of Micrococcus luteus Q24. Treatment groups 5–9 received Micrococcus luteus Q24 at doses of 10–10 cfu / mL, respectively. Thick horizontal bars indicate group means, error bars indicate 95% confidence intervals, and * indicates a significant difference at P<0.05. [Figure 7]Scatter plots of individual IL-6, IL-8, and IL-18 levels measured in conditioned medium of tissues on days 0 and 4 are shown. Thick horizontal bars are group means, and error bars indicate 95% confidence intervals. ** indicates significant differences at P<0.01. [Figure 8] Histology of EpiDerm treated with Micrococcus luteus Q24 for 5 days is shown (data not shown for PBS and Vitamin C). Sections were stained with hematoxylin and eosin (H&E) and imaged at 40x magnification. Arrow "B" points to the permeable membrane on which the tissue grew. The tissue layers shown are "C" stratum basale, "D" stratum granulosum, and "E" stratum corneum. The arrow labeled "A" indicates the remnants of a nucleus trapped in the stratum corneum (its presence may indicate accelerated stratum corneum production). [Figure 9] Scatter plots of individual changes in IL-6, IL-8, and IL-18 levels measured on days 4 and 5 in conditioned medium from tissues receiving the main treatment and the combination of ±0.5% sodium dodecyl sulfate (SDS). Thick horizontal bars represent group means, and error bars represent 95% confidence intervals. *** indicates a significant difference at P<0.001. [Figure 10] 1 shows the scores given by participants to various sensory attributes of balm formulations BB9, BB14, BB15 and BB18 in the sensory testing of the balms of Example 9. [Figure 11] 1 shows the overall scores of balms BB9, BB14, BB15 and BB18 in the sensory test of the balms of Example 9. [Figure 12] Figure 1 shows the percentage change in skin parameters analyzed using a DermoPrime skin analyzer (DermoPrime Viso, CHOWIS, South Korea) and associated software for study participants using Balm A (primary area / forearm, upper left; area of ​​concern, lower left) and Balm B (primary area / forearm, upper right; area of ​​concern, lower right) in the 7-day sensory and efficacy study of Example 10. [Figure 13]Figure 1 shows the percentage change in skin parameters at the area of ​​concern (Balm A) analyzed using the Dermo-Prime Viso skin analyzer and software in the sensory and efficacy study of Example 10. p-values ​​are calculated (95% confidence level) using a T-test (mean of two paired samples) for this data. [Figure 14] Figure 1 shows the percentage change in skin parameters at the area of ​​concern (Balm B) analyzed using the Dermo-Prime Viso skin analyzer and software in the sensory and efficacy study of Example 10. A p-value is calculated (95% confidence level) using a t-test (mean of two paired samples) for this data. [Figure 15] Percentage changes in skin moisture parameters (left) and keratin parameters (right) are shown. Participants were grouped by age into young (Group A, mean age 21-32 years) and middle-aged (38-50 years). Statistically significant differences were observed for both age groups, but for both balms applied to the areas of concern, more significant improvements (moisture content and keratin) were observed in the middle-aged group compared to the young group, suggesting an anti-aging effect of the balms. p-values ​​are calculated using a t-test (mean of two paired samples) at 95% confidence level. [Figure 16] Growth curves of Micrococcus luteus Q24 with prebiotic candidates are shown. [Figure 17] 1 shows growth curves for prebiotics—olive squalene and pomegranate seed oil combination; olive squalene and oatmeal flour (colloidal oatmeal) combination; and olive squalene and vitamin E combination. DETAILED DESCRIPTION OF THE INVENTION

[0089] 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 that includes the term "comprising," there may be features present other than the features preceding the term. Related terms such as "comprise," "comprised," and "comprises" should be interpreted similarly.

[0090] As used herein, the term "and / or" means "and" or "or," or both.

[0091] As used herein, "(s)" following a noun refers to the plural and / or singular form of the noun.

[0092] For example, general chemical and biological terms used in formulas herein have their ordinary meanings.

[0093] The terms "composition" and "formulation" are used interchangeably herein. As used herein, the term "balm" refers to a semi-solid topical composition that dissolves upon contact with the skin. Balm formulations of the present invention have a higher viscosity than serums or creams, with balm viscosities ranging from about 2,500,000 to about 8,000,000 cP at 25°C. As used herein, the term "balm" does not include hard lip balm sticks, which have viscosities higher than this range, typically exceeding 9,000,000 cP at 25°C.

[0094] The term "platform" is used herein to refer to a group of balm formulations having similar combinations of viscosity modifiers and oily bases.

[0095] As used herein, the term "subject" refers to mammals, including humans, dogs, cats, horses, sheep, cattle, and other domestic and farm animals.

[0096] Postbiotics are defined as preparations of inactivated microorganisms and / or their components that confer a health benefit to the host (Salminen, S., Collado, MC, Endo, A. et al. The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics. Nat Rev Gastroenterol Hepatol 18, 649-667 (2021)). Postbiotics suitable for use in the present invention are described below.

[0097] The term "self-emulsifying glyceryl monostearate" is used herein to refer to compositions that comply with the requirements of the BP monograph (2021) "Self-emulsifying Glyceryl Monostearate." IMWITOR® 960 K (IOI Oleo GmbH) is an ester of palmitic and stearic fatty acids with glycerol. The fatty acids and glycerol are derived from vegetable sources. It contains at least 30.0% monoglycerides and further contains approximately 5% alkaline stearates. IMWITOR® 960 K fully complies with the requirements of the current UK monograph "Self-emulsifying Glyceryl Monostearate."

[0098] 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. Determining colony forming units requires culturing the microorganism and counting only the viable cells on a nutrient agar medium, i.e., cells that can grow and form visible colonies.

[0099] The unit "% w / w" means a weight percentage based on the total weight of the composition.

[0100] As used herein, "room temperature" refers to a temperature of 25°C.

[0101] As used herein, the terms "lyoprotectant-free" or "cryoprotectant-free" mean that Micrococcus luteus was produced in the absence of a lyoprotectant or cryoprotectant, or both.

[0102] The term "preservative-free" is used herein to refer to compositions prepared without added preservatives.

[0103] The term "homogeneous" is used herein to refer to a composition that is homogeneous throughout. Homogeneity of the composition can be achieved by mixing the composition using various methods, such as manual stirring or automatic stirring or mechanical stirring, for example, using a magnetic stir bar or using a homogenizer.

[0104] As used herein, the term "improving the appearance of skin or at least one sign of aging in a subject" refers to improving at least one parameter commonly used in skin analysis, including skin radiance, skin health, skin hydration, pore size, skin softness, skin clarity, moisture level, sebum level, appearance of wrinkles, dryness, roughness, dullness, appearance of spots including age spots, and impurities.

[0105] Topical Compositions The present invention provides a topical composition comprising Micrococcus luteus Q24 and a viscosity adjusting agent, the topical composition having a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C.

[0106] The present invention also provides a topical composition comprising: Approximately 0.5-2% w / w of Micrococcus luteus Q24, about 15 to about 35% w / w beeswax, about 2 to about 10% w / w cetostearyl alcohol, about 10% to about 30% w / w cocoa butter, A sufficient amount of oily base, Including, The composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C. A topical composition is provided.

[0107] The present invention also provides a topical composition comprising: Approximately 1×10 3 ~Approx. 1×10 12 cfu / g Micrococcus luteus Q24, about 15 to about 35% w / w beeswax, about 2 to about 10% w / w cetostearyl alcohol, about 10% to about 30% w / w cocoa butter, A sufficient amount of oily base, Including, The composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C. A topical composition is provided.

[0108] The present invention also provides a topical composition comprising: Approximately 0.5-2% w / w of Micrococcus luteus Q24, about 10 to about 30% w / w beeswax, about 10% to about 30% w / w shea butter, about 10% to about 30% w / w cocoa butter, A sufficient amount of oily base, Including, The composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C. A topical composition is provided.

[0109] The present invention also provides a topical composition comprising: Approximately 1×10 3 ~Approx. 1×10 12 cfu / g Micrococcus luteus Q24, about 10 to about 30% w / w beeswax, about 10% to about 30% w / w shea butter, about 10% to about 30% w / w cocoa butter, A sufficient amount of oily base, Including, The composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C. A topical composition is provided.

[0110] In various embodiments, the composition further comprises about 0.5 to about 2% w / w polysorbate 80.

[0111] In various embodiments, the oil base is a medium chain triglyceride oil or olive oil.

[0112] In various embodiments, the composition does not contain preservatives.By way of background, preservatives typically used in prior art skin care products include antioxidants and antimicrobial agents, such as parabens, benzoates, sorbates, and sulfites.Some consumers have allergic reactions to preservative compounds or prefer to use natural products.Advantageously, the composition of the present invention does not require preservatives to prevent microbial contamination.

[0113] The topical compositions described herein can be used to improve the appearance of skin or at least one sign of aging. In various embodiments, the topical compositions can be used to improve skin hydration or reduce dryness of the skin.

[0114] Described herein is the use of a composition comprising Micrococcus luteus Q24 and a viscosity adjusting agent, the composition having a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C, in the manufacture of a medicament for improving the appearance of skin or at least one sign of aging.

[0115] Micrococcus luteus Q24 useful in the present invention has been found in tissue culture models to be well tolerated, not induce an anti-inflammatory response, have anti-inflammatory effects, promote the growth of a stratum corneum, which may aid in skin rejuvenation and hydration, and reduce skin pores and wrinkles (see Example 8). The Micrococcus luteus Q24 compositions described herein have also been shown to have beneficial effects on skin quality parameters, particularly increased moisture and reduced keratinization (see Example 10). Thus, the compositions described herein are also useful for improving the appearance of skin, including dry skin in infants, such as teething rash on the mouth, chin, neck, or chest due to saliva, and diaper rash due to feces or urine.

[0116] How to use Described herein is a method for improving skin appearance or at least one sign of aging, comprising applying to skin a topical composition comprising Micrococcus luteus Q24 and a viscosity adjusting agent, wherein the composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25° C. Also described herein is a topical composition comprising Micrococcus luteus Q24 and a viscosity adjusting agent for improving skin appearance or at least one sign of aging, wherein the topical composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25° C. Further described herein is the use of a topical composition comprising Micrococcus luteus Q24 and a viscosity adjusting agent, wherein the topical composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25° C., in the manufacture of a medicament for improving skin appearance or at least one sign of aging.

[0117] Micrococcus luteus Q24 Micrococcus luteus is a bacterial resident (commensal) of human skin and plays an important role in maintaining the balance between the various microbiota of the skin.

[0118] Micrococcus luteus Q24 was deposited with Deutsche Sammlung von Mikro organisms Und Zellkulturen GmbH, Braunschweig, Germany on March 10, 2005, and has been assigned accession number DSM 17172. Micrococcus luteus strain Q24 is described in WO 2006104403, which is incorporated herein by reference.

[0119] In various embodiments, Micrococcus luteus is a live probiotic.

[0120] In various embodiments, the compositions of the invention useful in the methods of the invention contain approximately 1 x 10 3 ~Approx. 1×10 12 In various embodiments, the composition comprises about 1 x 10 cfu / g of Micrococcus luteus Q24. 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 9In various embodiments, the composition comprises about 1 x 10 cfu / g of Micrococcus luteus Q24. 9 Contains cfu / g.

[0121] 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 probiotic-containing products 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 provides protection during drying, while a cryoprotectant provides protection during freezing. The same composition can have both functions, and these terms are used interchangeably herein unless otherwise specified.

[0122] Suitable lyoprotectants or cryoprotectants are known to those skilled in the art. In various embodiments, the lyoprotectant is trehalose or a ternary mixture comprising trehalose, maltodextrin, and lactitol.

[0123] Surprisingly, applicants have also determined that Micrococcus luteus Q24 can be freeze-dried (FD) in the absence of a lyoprotectant or cryoprotectant without significant loss of cell viability, as shown in Figure 1. This result is counterintuitive, as conventional methods require that microorganisms be protected during the freeze-drying process. In various embodiments, Micrococcus luteus Q24 does not include a lyoprotectant in the composition.

[0124] As can be seen, this finding provides significant manufacturing advantages in that the freeze-drying process is simplified and costs are reduced due to the elimination of the need for cryoprotectants or cryoprotectants.

[0125] Viscosity modifier The present composition contains one or more viscosity modifiers to achieve a viscosity in the range of about 2,500,000 to about 8,000,000 cP at 25°C. Compositions with viscosities in this range can be formulated as balm compositions. Such balm compositions allow live probiotics to persist on the skin for a long period of time. Balm formulations containing live Micrococcus luteus Q24 have not been described in the prior art. For example, the hard deodorant stick of WO 2006 / 104403 has a very high viscosity (greater than 9,375,000 cP at 25°C) that is outside this range and beyond the capabilities of the measuring equipment used by the inventors. Although not measured, the saline suspension of WO 2006 / 104403 is expected to have a viscosity comparable to that of water (approximately 1 cP at 25°C).

[0126] In various embodiments, the composition comprises one or more viscosity modifiers in a total amount of about 30 to about 80% w / w. For example, the composition may comprise the viscosity modifier(s) in an amount of about 30 to about 70%, or about 30 to about 60%, or about 30 to about 55%, or about 40 to about 80%, or about 40 to about 70%, or about 40 to about 60%, or about 40 to about 55%, or about 45 to about 55% w / w. For example, the composition may contain about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, about 123%, about 124%, about 12 %, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, or about 80% w / w. In various embodiments, the composition comprises one or more viscosity modifiers (e.g., a combination of white wax or yellow wax with cetostearyl alcohol) in an amount of about 50% w / w.

[0127] Suitable viscosity modifiers include, but are not limited to, hydrophobic silica, hydrophilic silica, white wax, yellow wax, paraffin wax, jojoba wax, microcrystalline wax, ethylcellulose, emulsifying wax, stearic acid, xanthan gum, tapioca starch, Carbopol polymers (e.g., 971p, 974p), cocoa butter, shea butter, fatty alcohols, self-emulsifying glyceryl monostearate, candelilla wax, lanolin, and combinations of any two or more thereof. In a preferred embodiment, the viscosity modifier is selected from a combination of white wax or yellow wax with cetostearyl alcohol.

[0128] In some embodiments, the viscosity modifier comprises a fatty alcohol, such as cetyl alcohol, stearyl alcohol, or cetostearyl alcohol. In a preferred embodiment, the viscosity modifier comprises cetostearyl alcohol. Cetostearyl alcohol, also known as cetearyl alcohol or cetylstearyl alcohol, is a mixture of the fatty alcohols cetyl alcohol and stearyl alcohol. The inventors have found that balm formulations containing cetostearyl alcohol exhibit good spreadability and sensory properties, as further described below.

[0129] In some embodiments, the viscosity modifier comprises a combination of self-emulsifying glyceryl monostearate, shea butter, and cocoa butter. In various embodiments, the composition comprises self-emulsifying glyceryl monostearate, shea butter, and cocoa butter in a combined amount of about 30 to about 80% w / w, e.g., about 30 to about 70%, or about 40 to about 60%, or about 45 to about 55% w / w. In various embodiments, the composition comprises self-emulsifying glyceryl monostearate in an amount of about 5 to about 30% w / w, shea butter in an amount of about 10 to about 35% w / w, and cocoa butter in an amount of about 10 to about 35% w / w.

[0130] In some embodiments, the viscosity modifier comprises a combination of white or yellow wax, cetostearyl alcohol, and cocoa butter. In various embodiments, the composition comprises white or yellow wax, cetostearyl alcohol, and cocoa butter in a total amount of about 30 to about 80% w / w, e.g., about 30 to about 70%, or about 40 to about 60%, or about 45 to about 55% w / w. In various embodiments, the composition comprises white or yellow wax in an amount of about 15 to about 35% w / w, cetostearyl alcohol in an amount of about 2 to about 10% w / w, and cocoa butter in an amount of about 10% to about 30% w / w.

[0131] In some embodiments, the viscosity modifier comprises a combination of white or yellow wax, shea butter, and cocoa butter. In various embodiments, the composition comprises white or yellow wax, shea butter, and cocoa butter in a total amount of about 30 to about 80% w / w, e.g., about 30 to about 70%, or about 40 to about 60%, or about 45 to about 55% w / w. In various embodiments, the composition comprises white or yellow wax in an amount of about 15 to about 35% w / w, shea butter in an amount of about 10% to about 30% w / w, and cocoa butter in an amount of about 10% to about 30% w / w.

[0132] 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. In addition, the dispersing agent can aid in emulsifying the oil and aqueous phases when they are mixed together prior to application. The dispersing agent can be a nonionic dispersing agent or an amphoteric dispersing agent. Examples of nonionic 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), and cetearyl olivate. Examples of amphoteric dispersing agents include, but are not limited to, lecithins such as egg lecithin and soybean lecithin, sodium stearoyl glutamate, and sodium cocoyl isethionate.

[0133] 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.

[0134] oily base In various embodiments, the composition comprises an oily base. Suitable oily bases include, but are not limited to, medium-chain triglycerides (MCT) and vegetable oils. Preferably, the medium-chain triglyceride is caprylic / capric triglyceride, such as Miglyol 812N (a triglyceride ester of caprylic and capric fatty acids derived from saturated coconut oil / palm kernel oil with vegetable-derived glycerol). In various embodiments, the vegetable oil is selected from the group consisting of sunflower oil, canola oil, soybean oil, olive oil, jojoba oil, argan oil, rosehip oil, marula oil, chamomile oil, tamanu oil, grapeseed oil, calendula oil, pomegranate oil, macadamia oil, barberry fruit oil (e.g., available from Paris Fragrances (USA)), sweet almond oil, evening primrose oil, and any combination of two or more thereof. In some embodiments, the oil is MCT oil, olive oil, or a combination thereof.

[0135] In various embodiments, the composition comprises an oily base in a sufficient quantity (qs), i.e., an amount that brings the total % w / w of the composition to 100%. In various embodiments, the composition comprises an oily base in an amount of about 20 to about 70% w / w. For example, the composition may comprise an oily base in an amount of about 30 to about 70%, or about 30 to about 60%, or about 30 to about 55%, or about 40 to about 70%, or about 40 to about 60%, or about 40 to about 55%, or about 45 to about 55% w / w. For example, the composition can comprise an oily base in an amount of about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, or about 70% w / w. In various embodiments, the composition comprises an oily base in an amount of 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 63% w / w.

[0136] The combination of viscosity modifier(s) and oily base described herein advantageously provides a balm formulation suitable for maintaining the microbiological stability and viability of Micrococcus luteus Q24 and for providing good temporal, thermal and physical stability of the formulation.

[0137] 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 compositions, water includes moisture absorbed from the environment.

[0138] 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), and the like. plantarum), Bifidobacterium species (e.g., B. bifidum, B. longum, or B. lactis 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).

[0139] S. salivarius K12 was deposited on October 8, 1999, with the Deutsche Sammlung von Mikro organismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124, Braunschweig, Germany, and has been assigned the accession number DSM 13084. S. salivarius M18 was deposited on December 12, 2001, with the Deutsche Sammlung von Mikro organismen und Zellkulturen GmbH, Mascheroder Weg 1 b, D-38124, Braunschweig, Germany, and has been assigned the accession number DSM 14685.

[0140] 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 24 SMB, 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 24 SMB, Streptococcus oralis (e.g., S. oralis 89a), Streptococcus salivarius DB-B5, and any combination of two or more thereof.

[0141] In various embodiments, the composition contains about 1 x 10 each additional probiotic. 3 ~Approx. 1×10 12 For example, the composition may contain each additional probiotic in an amount of about 1 x 10 cfu / g. 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 In various embodiments, the composition contains about 1 x 10 cfu / g of each additional probiotic. 9 Contains cfu / g.

[0142] Postbiotics In various embodiments, the composition comprises a postbiotic. Postbiotics suitable for use in the present invention include fermentates, filtrates, lysates, and supernatants. In various embodiments, the postbiotic is selected from the group consisting of a fermentate, filtrate, supernatant, fermentation filtrate, or lysate selected from the group consisting of a Streptococcus fermentate, a Streptococcus fermentation filtrate, a Streptococcus fermentation lysate, a Streptococcus lysate, a Streptococcus filtrate, a Lactobacillus (including Rimosilactobacillus spp., Ligilactobacillus spp., and Lactiprancylactibacillus spp.) fermentate, a Lactobacillus (including Rimosilactobacillus spp., Ligilactobacillus spp., and Lactiprancylactibacillus spp.) filtrate, a Micrococcus fermentation lysate, a Bifidobacterium fermentation lysate, a Bifidobacterium fermentation filtrate, a Galactomyces fermentation filtrate, a Saccharomyces fermentation filtrate, a Bacillus fermentate, a Bacillus filtrate, and a combination of any two or more thereof. The postbiotic may be in any suitable form, including a liquid or a powder, for example, a freeze-dried or spray-dried powder.

[0143] In various embodiments, the composition comprises from about 0.1% to about 35% w / w postbiotic, for example, from about 10% to about 15% w / w postbiotic.

[0144] In various embodiments, the composition comprises the liquid postbiotic in an amount of about 0.1% to about 30% w / w, for example, about 10% to about 15% w / w.

[0145] In various embodiments, the composition comprises the powdered postbiotic in an amount of about 0.1% to about 20%, for example, about 10% to about 15% w / w.

[0146] Additional additives Those skilled in the art will understand that topical compositions may contain other additives conventionally used in topical compositions, such as moisturizers. Those skilled in the art will further understand that additives must balance the viability and efficacy of the probiotic. 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, buffering agents, antimicrobial agents, prebiotics, fragrances, antioxidants, colorants, skin protectants, antimicrobial agents, aluminum salts, inorganic 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 understand that any additional additives should not be inhibitory to Micrococcus luteus Q24.

[0147] Those skilled in the art will also appreciate that some additives may have dual functionality, for example, an antioxidant or skin protectant may also function as a prebiotic.

[0148] In various embodiments, the composition further comprises an inhibitory activity enhancer, a buffering agent, an antimicrobial agent, a prebiotic, a postbiotic, a fragrance, an antioxidant, a colorant, a skin protectant, an antimicrobial agent, an aluminum salt, an inorganic pigment, an odor absorber or neutralizer, a sunscreen, and combinations of any two or more thereof.

[0149] 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.

[0150] 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.

[0151] In various embodiments, the antimicrobial 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 antimicrobial 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.

[0152] In various embodiments, the composition includes a prebiotic, which can enhance the growth of Q24, enhance antimicrobial activity, or enhance the production of compounds that enhance skin quality parameters.

[0153] In various embodiments, the prebiotic is selected from the group consisting of manuka honey, olive squalene, 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, inulin (e.g., inulin from Chichorium intybus (chicory) root), Borago officinalis (borage) seed oil, pomegranate (Punica granatum) extract, Siraitia grosvenorii (Sarah fruit) extract, colloidal oatmeal, xylitol, yeast extract, fructooligosaccharide powder, fructooligosaccharide liquid, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and combinations of any two or more thereof.

[0154] In various embodiments, the prebiotic is selected from the group consisting of yeast extract, cysteine, maltodextrin, inulin (e.g., inulin from chicory root), 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 (e.g., inulin from chicory root), licorice root, licorice extract, and combinations of any two or more thereof.

[0155] In various embodiments, the prebiotic may be an oil prebiotic or a powder prebiotic. In various embodiments, the composition comprises an oil prebiotic and / or a powder prebiotic. In various embodiments, the oil prebiotic is selected from the group consisting of olive squalene, pomegranate seed oil, flaxseed oil, coconut oil, vitamin E, olive oil, calendula oil, almond oil, tomato oil, fructooligosaccharide liquid, and combinations of any two or more thereof. In various embodiments, the powder 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, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and combinations of any two or more thereof.

[0156] In various embodiments, the prebiotic is a combination of olive squalene and pomegranate seed oil; a combination of olive squalene and vitamin E; or a combination of olive squalene, pomegranate seed oil, and vitamin E. The inventors have surprisingly determined that the combination of olive squalene and pomegranate seed oil or the combination of olive squalene and vitamin E has a synergistic effect on the growth of Micrococcus luteus Q24.

[0157] In various embodiments, the composition comprises from about 0.1% to about 10% w / w prebiotic, e.g., 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 prebiotic.

[0158] 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 from 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% to about 25%, or contains 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 prebiotics.

[0159] 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 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% 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% oil prebiotics.

[0160] In various embodiments, the composition comprises a powdered prebiotic in an amount of 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 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 contains 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 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 powdered prebiotic.

[0161] 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 squalene. 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 squalene. 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 1.2%, or about 1% w / w vitamin E.

[0162] The present inventors have unexpectedly determined that the combination of the prebiotics olive squalene and pomegranate seed oil; and the combination of the prebiotics olive squalene and vitamin E are synergistic. Thus, in certain embodiments, the composition comprises about 0.1% to about 10% w / w olive squalene, 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 squalene, 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 squalene, about 3% to about 8% w / w olive squalene, or about 4 to about 7% w / w olive squalene, for example, about 5% w / w olive squalene, 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 1.2%, or about 1% w / w vitamin E.

[0163] In certain embodiments, the composition comprises about 0.1% to about 10% w / w olive squalene, 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 squalene; 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; 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 1.2%, or about 1% w / w vitamin E.

[0164] 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 may be added as an oil or a powder.

[0165] 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, bakuchiol, licorice root, and combinations of any two or more thereof. In various embodiments, the antioxidant is selected from the group consisting of vitamin E, resveratrol, squalene (e.g., olive squalene), vitamin C, beta-carotene, retinyl acetate, retinyl palmitate, retinol, niacinamide, pomegranate seed oil, flaxseed oil, and combinations of any two or more thereof.

[0166] 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, palmitic acid heptapeptide-15, 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 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 may be 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, palmitic acid heptapeptide-15, 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 extract, silicone, xylitol, oat extract, oatmeal powder, colloidal oatmeal, aloe vera, sunscreen, lactic acid, Camellia oleifera (camellia) oil, e.g., Goat's milk, Native (NZ), Cannabis Sativa (Hemp) Seed Oil, Hippophae (Sea Buckthorn) Fruit Oil, and combinations of any two or more thereof.

[0167] 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, tea tree oil, salicylic acid, and combinations thereof.

[0168] In various embodiments, the aluminum salt is an aluminum chlorohydrate salt or other salt commonly used in antiperspirants.

[0169] In various embodiments, the inorganic pigment is zinc oxide and / or titanium dioxide. Inorganic pigments are known to protect against ultraviolet radiation (e.g., from the sun).

[0170] In various embodiments, the odor absorber or neutralizer is zinc ricinoleate or sodium lauroyl sarcosinate.

[0171] In various embodiments, the sunscreen is oxybenzone, avobenzone, octisalate, octocrylene, homosalate, or octinoxate. Inorganic sunscreens use zinc oxide and / or titanium dioxide.

[0172] Those skilled in the art will also understand that when the composition is applied in combination with an aqueous phase, the additional additives described may also be provided in the aqueous phase.

[0173] 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.Improving the appearance of skin or the signs of aging can include, but are not limited to, effects such as skin looking brighter, skin looking healthier, skin feeling more hydrated, pores becoming smaller, skin feeling softer, skin looking clearer, increased moisture, reduced sebum production, and reduced appearance of wrinkles, dryness, roughness, dullness, age spots (including age spots), and impurities.

[0174] The compositions described herein are also useful for improving the appearance of skin, including dry skin, in infants. Acidification in an infant's body fluids can be caused by acidic saliva and changes in the gastrointestinal tract. This can lead to dry skin problems in infants, including teething rash on the mouth, chin, neck, or chest due to saliva, and diaper rash due to feces or urine.

[0175] Pore ​​size, skin moisture content, sebum production, wrinkles, age spots, dead skin cells, and impurities can all be measured using a skin analyzer (e.g., Dermo Prime Viso, CHOWIS, South Korea). Equipped with artificial intelligence, the skin analyzer quantitatively measures changes in various skin parameters, such as moisture retention, sebum, pores, wrinkles, age spots / pigmentation, impurities, dead skin cells, skin tone, dark spots, and skin sensitivity. The device is equipped with a moisture sensor and utilizes advanced optical technology with interchangeable lenses to measure up to 10 different skin measurements. It performs precise analysis of high-resolution images via the DermoBella app installed on an Android (Google Inc.) or iOS (Apple) tablet or smartphone. To test dead skin cells, exfoliating strips (Chowis, South Korea) may be used in conjunction with the skin analyzer. The exfoliating strips can be analyzed using the skin analyzer by placing them adhesive-side down on the skin, applying light pressure, leaving them on for 10 seconds, and then peeling them off. The device is used according to the protocol described in the Examples section of this specification. Skin parameter measurements are performed on the same skin area each time.

[0176] The degree of impurities such as redness can be analyzed by measuring the amount of porphyrin, which appears as a scarlet or orange glow in response to a specific wavelength range of UV light.

[0177] Impurities appear as either scarlet or yellow-green. While these can be analyzed separately, the device combines the two and classifies them as impurities. The index is calculated as a percentage of the image size. There is no arbitrary value for classifying the degree of severity of redness based on the amount of porphyrin detected.

[0178] Topical Composition Form In various embodiments, the composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25° C., e.g., about 3,000,000 to about 7,500,000, about 3,000,000 to about 7,000,000, about 3,500,000 to about 7,500,000, about 3,500,000 to about 7,000,000, or about 3,600,000 to about 7,000,000 About 3,700,000 to about 7,000,000, about 3,800,000 to about 7,000,000, about 3,900,000 to about 7,000,000, about 4,000,000 to about 7,000,000, about 4,000,000 to about 6,900,000, about 4,000,000 to about 6,800,000, about 4,000,000 to about 6 ,700,000, about 4,000,000 to about 6,600,000, about 4,000,000 to about 6,500,000, about 4,000,000 to about 6,400,000, about 4,000,000 to about 6,300,000, about 4,000,000 to about 6,200,000, about 4,000,000 to about 6,100,000, about 4,0 00,000 to about 6,000,000, about 4,000,000 to about 5,000,000, about 4,100,000 to about 7,000,000, about 4,200,000 to about 7,000,000, about 4,300,000 to about 7,000,000, about 4,400,000 to about 7,000,000, about 4,500,000 to about 7,000 ,000, about 4,500,000 to about 6,000,000, about 4,500,000 to about 6,900,000, about 4,500,000 to about 6,800,000, about 4,500,000 to about 6,700,000, about 4,500,000 to about 6,600,000, about 4,500,000 to about 6,500,000 cP. In various embodiments, the composition has a viscosity of about 4,500,000, 4,600,000, 4,700,000, 4,800,000, 4,900,000, 5,000,000, 5,100,000, 5,200,000, 5,300,000, 5,400,000, 5,500,000, 5,600,000, 5,700,000, 5,800,000, 5,900,000, 6,000,000, 6,100,000, 6,200,000, 6,300,000, 6,400,000, or 6,500,000 cP at 25°C.

[0179] Viscosity can be measured at 25° C. using a Brookfield LVDVI Prime with a Brookfield Helipath Spindle (S94 or S95 or S96) set at 0.5 RPM. Those skilled in the art will understand that other methods can be used to measure viscosity and that viscosity measurements can vary depending on the method used.

[0180] In various embodiments, the composition has a shelf life of at least 3 months at 25° C. and 60% RH (relative humidity). In various embodiments, the composition has a shelf life of at least 6 months, at least 12 months, or at least 24 months at 25° C. and 60% RH (relative humidity).

[0181] Methods for preparing topical compositions In one aspect, the present invention provides a method for preparing a topical composition comprising Micrococcus luteus Q24, the method comprising: a) heating a mixture of an oily base and a viscosity modifier to provide a molten mixture; b) cooling the molten mixture to a temperature in the range of 38°C to 28°C; c) adding Micrococcus luteus Q24 to the molten mixture from step b) to provide a homogenous mixture; d) cooling the mixture from step c) to room temperature to provide the composition. The present invention provides a method of manufacturing a semiconductor device, comprising:

[0182] In some embodiments, the dispersant is added during step (a) or before step (b).

[0183] Micrococcus luteus Q24 has been found to be heat-sensitive at high temperatures (>40°C). Heating impairs cell viability. The viscosity modifiers used in the present invention are solid or semi-solid at room temperature and are typically processed at high temperatures. These solid viscosity modifiers pose unique challenges in the preparation of compositions containing live, viable microorganisms, such as Micrococcus luteus Q24. After much trial and effort, the applicants have developed a method for preparing the composition that allows for efficient production of the composition while maintaining optimal viability of the probiotic.

[0184] In various embodiments, the molten mixture is cooled to a temperature in the range of about 35 to about 28°C, e.g., about 35 to about 29°C, about 35 to about 30°C, about 35 to about 31°C, about 34 to about 28°C, about 34 to about 29°C, about 34 to about 30°C, about 33 to about 30°C, about 32 to about 29°C, or about 31 to about 29°C. In various embodiments, the molten mixture is cooled to a temperature of about 35, 34, 33, 32, 31, 30, 29, or 28°C. In some embodiments, cooling can be accomplished by simply removing the molten mixture from a heat source, such as a hot plate used in heating step (a). In some embodiments, the molten mixture can be stirred while cooling, for example, by placing it on a magnetic stir plate, and once the molten mixture has cooled to the temperature required for step (b), Micrococcus luteus Q24 can be added.

[0185] In embodiments where the Micrococcus luteus Q24 raw material component is added to the formulation at an elevated temperature, it may be necessary to maintain the formulation at that temperature, which may limit the ability to scale up the process to produce larger batches. To address this issue, Applicants have worked to achieve a balance between cooling and the timing of addition of Micrococcus luteus.

[0186] In one aspect, the present invention provides a method for preparing a topical composition comprising Micrococcus luteus Q24, the method comprising: a) heating a mixture of an oily base and a viscosity modifier to provide a molten mixture; b) cooling the molten mixture to a temperature of about 22°C to about 27°C; c) adding Micrococcus luteus Q24 to the cooled mixture from step b); d) mixing to provide a uniform mixture. The present invention provides a method of manufacturing a semiconductor device, comprising:

[0187] In various embodiments, the mixing in step (d) can be accomplished using a high-shear homogenizer or overhead stirrer. In various embodiments, the molten mixture is cooled to room temperature in step (b). Thus, the method advantageously does not include heating the Micrococcus luteus Q24 above room temperature, thereby enhancing the microbiological stability of the live probiotic. An additional advantage is that there is no need to control the addition of Micrococcus luteus Q24 raw material ingredients during the cooling step, providing advantages for large-scale batch production.

[0188] Thus, the topical compositions described herein can be prepared by first mixing an oily base with a viscosity modifier, adding Micrococcus luteus Q24 to the mixture, and homogenizing the mixture (e.g., for about 1 to 3 minutes) to obtain the composition. In various embodiments, the mixture is homogenized using a high-shear homogenizer or an overhead stirrer.

[0189] Micrococcus luteus Q24 can be milled 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 milling and sieving techniques will be apparent to those skilled in the art. For example, Micrococcus luteus Q24 can be milled using a dry powder mill (e.g., Quadro Powder Mills, e.g., U10 or U21). A smaller particle size can be advantageous in providing improved spreadability of the composition, enhancing dispersibility when combined with an aqueous phase, and providing improved sensory properties of the composition (i.e., not being coarse or gritty).

[0190] 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 a laser diffraction method for particle size analysis of dry powder dispersions (Malvern Instruments, USA). One skilled in the art will understand that particles with a Dv90 of less than 300 μm can pass completely through a 250 μm sieve.

[0191] The stability of the balm formulations of the present invention can be examined using standard tests for stability with respect to time, temperature, humidity, and physical force (syneresis). Syneresis is the separation of the liquid phase from the solid phase of a balm / gel-based system due to matrix shrinkage over time. This phenomenon is a slow process under normal conditions of real-time storage. Syneresis is undesirable in consumer products such as balm formulations. The physical appearance and uniformity of dosage and sensory properties can be significantly affected by syneresis. Wax systems tend to have high physical stability due to strong wax-crystal bonding interactions that cause lattice formation (Ivanovszky, L. Waxes: "Colloidal properties and systems", Journal of Polymer Science, 58(166), 273-288, 1962). The addition of additional ingredients such as oils and butters disrupts the lattice structure, thereby changing the elasticity of the wax against external forces. Syneresis occurs when the formulation structure is disrupted, resulting in oil leakage.

[0192] A low tendency to syneresis (e.g., a composition exhibiting less than 15% syneresis at 13,000 rpm) improves the sensory properties of a skin composition, as the composition is easier to spread and absorb into the skin. This can also be useful for analytical testing, as it makes it easier to sample and mix the product. However, a high tendency to syneresis (e.g., greater than 20% syneresis at 13,000 rpm) can change product properties over time. This inconsistency and lack of physical stability is not ideal for a commercial skin care formulation, and furthermore, it is likely to affect the stability of live cultures present in the formulation.

[0193] How to use The present invention also relates to a method for 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 may be used as a balm. The composition may be applied in an amount appropriate to address the subject's skin concerns. Those skilled in the art will understand that the composition may be applied to subjects of any age, from the very young, including infants, to the very elderly. The amount applied may vary depending on the subject's age, sex, and the concerns being addressed. A typical application regimen may include applying the composition of the present invention monthly, weekly, daily, or 1 to 4 times daily.

[0194] The composition can be applied by hand from a bottle or other container or with an applicator. The amount applied can be selected by the user depending on the area to be treated. The composition can be applied to the area of ​​concern (e.g., dry areas such as elbows, knees, etc.) or more broadly to any part of the body.

[0195] The compositions of the present invention can be used in any daily skin care regimen in the same manner as conventional skin balms. A typical regimen may involve using the compositions of the present invention once or twice daily. Long-term or limited-term use of the compositions, for example, for 1 to 2 months, is contemplated. [Example]

[0196] material Equipment: Unless otherwise stated, the components of the balm formulations were mixed using a magnetic stirrer and hot plate (DIAB MS-H280-Pro). Samples for recovery analysis were mixed using a stomacher (Masticator Basic IUL Instruments, New Zealand). Culture colonies were counted using a Q Count Automated Colony Counter (Hunting Tree Bioscience Supplies, New Zealand).

[0197] Chemicals: Medium chain triglycerides (caprylic / capric triglycerides), such as Miglyol 812 (European Pharmacopoeia grade) or Radia 7104, were purchased from Sasol, Hamburg, Germany or Oleon, Malaysia, respectively. Tween® 80 (European Pharmacopoeia grade) was purchased from Sigma (New Zealand). Hydrophobic silica (Aerosil R 972 (pharmaceutical grade) was purchased from Evonik, Germany (supplied by Chemiplas, Auckland, New Zealand). Beeswax, cetostearyl alcohol, cocoa butter, shea butter, pomegranate seed oil, vitamin E, olive squalene, colloidal oatmeal USP were purchased from Pure Ingredients Ltd (New Zealand). Candelilla wax was purchased from Go Native (New Zealand). Emulsifying wax was purchased from Lotus Oils Ltd (New Zealand). Self-emulsifying glyceryl monostearate (Imwitor® 960K) was purchased from IOI Oleo (Germany). Ethyl cellulose was purchased from Colorcon (UK). Olive oil was purchased from Lipoid (Germany). Distilled water was used for sample and culture preparation. All other chemicals and solvents were of analytical grade (Sigma, New Zealand).

[0198] The active probiotic, Micrococcus luteus Q24 lyophilized raw component powder, was produced by Blis Technologies. Recovery assays and dilutions were prepared using analytical-grade phosphate-buffered saline (PBS) solution (Oxoid, Dulbecco's A) purchased from Thermofisher. Sample dispersions were prepared using a non-filter stomacher bag (BagLight PolySilk Transparent, 400 mL) and a filter-equipped stomacher bag (BagPage+ Full-page filter bag with Microperforated filter, 400 mL) (both purchased from Interscience, New Zealand). Bacterial cultures were grown on CABK12, sheep, and human blood agar plates purchased from Fort Richard Laboratories, New Zealand. Deionized water was used to prepare buffers and agar media.

[0199] Instead of purchasing CAB K12 agar (Columbia blood agar) plates, they may be prepared using the following ingredients: 44.0 g Columbia blood agar base (bd.com, USA), 5.0 g yeast extract (Bacto, bd.com, USA), 2.5 g glucose, and 1.0 g calcium carbonate (Sigma-Aldrich, USA); or other amounts of these ingredients in equivalent weight ratios. A suitable procedure is as follows: CAB K12 agar is suspended in 1 L of distilled water in an appropriately sized conical flask or beaker (large enough to prevent boiling over). The flask or beaker is covered with aluminum foil and heated on a hot plate with stirring until boiling. Boil for 1 minute to completely dissolve the ingredients, then autoclave the solution at 121°C for 15 minutes. Cool to 50°C, mix thoroughly to evenly suspend the calcium carbonate, adjust the pH to 7.3±0.2 if necessary, and aseptically place in a Petri dish.

[0200] The minimum inhibitory concentrations (MICs) of formulation components against Micrococcus luteus Q24 were tested by spreading a Micrococcus luteus Q24 lyophilized raw component suspension, prepared by adding 3–5 Micrococcus luteus Q24 colonies (from cultures grown overnight on sheep blood agar plates) to 3 mL of PBS, onto CABK12 agar plates. The components were serially diluted 1:10 in an oil base to yield concentrations of 5%, 1.25%, 0.625%, and 0.156% v / v. Finally, 20 μL of each concentration was spotted onto a segment of an agar plate (100%, 10%, 5%, 1.25%, 0.625%, and 0.156%). The plates were incubated at 37°C in 5% CO2 for 24 hours. After incubation, the presence of a zone of inhibition was recorded to determine the MIC. The test results confirmed that the ingredients used in the balm formulations shown in Table 1 had no inhibitory activity against Micrococcus luteus Q24. Examples 1-4 - Preparation of balm formulations Balm formulations were prepared having the ingredients shown in Table 1. Once the balm formulations were prepared, they were stored at room temperature or refrigerated conditions for at least 3 days before physicochemical or sensory evaluation. [Table 1] [Table 2]

[0201] Example 1: Wax-based balm formulation Formulations were prepared having the waxes shown in Table 2 and other ingredients shown in Table 1. [Table 3]

[0202] A 100 mL glass beaker was placed inside another beaker filled with water to create a jacketed water bath. The wax in the 100 mL glass beaker was heated on a magnetic stirrer hot plate until it reached the temperature (45°C-70°C) specified for each wax (Table 2). The formulation temperature was measured using an infrared thermometer gun or temperature probe. Once the wax was completely melted (a process that took approximately 10 minutes), the other ingredients listed in Table 1 were slowly added to the molten wax with continuous stirring (600-700 rpm) using a magnetic stirrer. Once the mixture was completely dissolved (clear solution, approximately 10 minutes), the heat was turned off and the beaker was transferred to another (unheated) stir plate. Stirring was continued and the mixture was allowed to cool slowly.

[0203] Once the temperature reached 30 °C, Micrococcus luteus Q24 powder was slowly added to the homogeneous mixture. To ensure homogeneity, mixing was achieved using a magnetic stirrer or by manual mixing with a glass rod.

[0204] Example 2: Wax-based balm formulation; cold process The live Micrococcus luteus Q24 raw material component is temperature sensitive. Therefore, we sought an alternative approach to the melting method of Example 1. In this alternative method, Micrococcus luteus Q24 was added once the formulation had cooled to room temperature. Due to its viscosity at this temperature, a high-shear homogenizer was used in this example.

[0205] Formulation BLT-BB9 was prepared according to Example 1 above, using the ingredients in the proportions listed in Table 1. The corresponding formulation, BLT-BB9B (Table 1), was prepared by modifying the method of Example 1 by adding Micrococcus luteus Q24 powder after the formulation had cooled to room temperature, rather than after the temperature reached 30°C. The formulation was then mixed using a high-shear homogenizer (DLAB D-160 Ultra-Turrax, IKA Instruments, Germany (via Lab Supply, New Zealand)) for a period of time to avoid the addition of heat due to friction. The homogenizer was operated for 3-5 minutes, gradually increasing the speed from approximately 8,000 rpm (speed 1 on the instrument dial) to a maximum of approximately 18,000 rpm (speed 4), and then decreasing it to approximately 8,000 rpm.

[0206] Cell counts for formulations BB9 and BB9B were obtained at weeks T0 and T2 and compared with their corresponding counts. The sensory properties of these balms prepared using different techniques were also compared.

[0207] No statistically significant difference in cell counts between the two samples was observed. Based on these preliminary results, it can be concluded that the use of a homogenizer does not affect the cell count of the product. Informal sensory evaluation suggested little difference in the applied sensory properties, but the visual appearance of formulation BLT-BB9B of Example 2 was preferable to formulation BLT-BB9 of Example 1. Example 3: Ethylcellulose-based balm formulation The following example was used to prepare formulation BLT-BB21, shown in Table 1. In a 100 mL glass beaker, oil was heated on a magnetic stirrer hot plate until a temperature of 160-170 °C was reached (measured using an infrared thermometer gun or temperature probe). Ethyl cellulose was slowly added to the heated oil while continuously stirring (600-700 rpm) with a magnetic stir bar. A stirring speed of 600-700 rpm was used to achieve good solubilization of the ethyl cellulose; higher speeds were found to result in the formation of air bubbles. To avoid the formation of ethyl cellulose lumps, the ethyl cellulose was added slowly to the oil, and continuous stirring was used. Once the ethyl cellulose was completely dissolved (clear solution, approximately 40 minutes), the heat was turned off and the beaker was transferred to another (unheated) stir plate. The ethyl cellulose-oil mixture was allowed to cool slowly while continuing to stir. When the temperature reached 60-70 °C, polysorbate 80 was added, followed by wax with continuous stirring until a homogeneous mixture was obtained. Once the temperature reached 30 °C, Micrococcus luteus Q24 powder was slowly added to the mixture.

[0208] Example 4: Balm formulation containing silica Balm formulations containing silica were produced following the same methodology as the balm formulations of Examples 1 and 3, with the silica added once the formulation had cooled to room temperature. For Example 2, the silica was blended with the cooled formulation using a high shear homogenizer for 3-5 minutes, gradually increasing the speed on the machine's speed dial from 1 to 4, then decreasing from 4 to 1.

[0209] Micrococcus luteus Q24 was used for all microbiological test samples and pilot efficacy studies.

[0210] Example 5: Stability Study - Over Time The viability of Micrococcus luteus Q24 in the balm formulations was evaluated under different storage conditions of temperature and humidity according to the ICH guideline for stability testing (ICH Q1AR2). 150 g batches of each of the six balm formulations, BLT-BB14, BLT-BB15, BLT-BB23, BLT-BB24, BLT-BB25, and BLT-BB26, were prepared in glass beakers (250 mL) according to the procedure described in Example 1. [Table 4]

[0211] The ingredients of these formulations are listed in Table 1.

[0212] The balm formulations were stored in 30 mL glass containers with thin plastic seals and plastic twist-on lids. 15 g of each balm formulation was filled into glass containers and stored in real time (25°C ± 2°C / 60% RH). The results are shown in Figure 2.

[0213] For comparison, a deodorant stick formulation was made according to the process described in Example 2 of WO 2006 / 104403.

[0214] Figure 3 shows the microbial cell counts of the deodorant stick disclosed in WO 2006 / 104403 under controlled storage conditions (25°C / 60% RH). The hard deodorant stick formulation of WO 2006 / 104403 contains 92% w / w shea butter / cocoa butter and has a very high viscosity (>9,375,000 cP), making it difficult to spread and therefore unable to be applied to the skin as a balm. The deodorant stick formulation of WO 2006 / 104403 is formed at a high melting temperature (>40°C), at which point the addition of Micrococcus luteus Q24 would be detrimental to long-term storage. The high heat weakens the cells, and although they remain viable at the initial stability point (1 month), they rapidly decline thereafter, indicating a lack of long-term shelf life. A decrease in cell counts is observed within one month compared to the balm formulation of the present invention, which maintains viability for at least three months.

[0215] Example 6: Viscosity and spreadability viscosity The viscosity of several different formulations was measured using a Brookfield LVDV1-Prime with Brookfield Helipath Spindles (S94, S95, or S96), and the results are shown in Table 4. [Table 5] Spindle and speed (a) S95, 1 rpm (b) S94, 0.5 rpm (c) S95, 0.5 rpm (d) S96, 0.5 rpm (e) S95, 0.5 rpm (f) S96, 0.1 rpm (g) S96, 0.1 rpm.

[0216] The serum formulations were measured to have an average viscosity of 144,000 to 1,410,000 cP, with viscosity increasing with silica content (Table 6). Formulations BB15 and BB25 were measured to have an average viscosity of 4,615,000 and 4,871,300 cP, respectively. The viscosity of the deodorant stick formulation of Example 2 of WO 2006 / 104403 was so high that it was outside the measurement range of the equipment used (maximum measurable viscosity of 9,375,000 cP) (Table 4).

[0217] Comparative Observations and Tests - Viscosity; Microbiological Stability A grape seed oil suspension was made using Micrococcus luteus Q24 raw material with a particle size of 290 μm (Dv90) according to the process described in WO 2006 / 104403, Example 3. The formulation was low viscosity and flowable due to uneven distribution of the raw material, gritty due to uneven particle distribution, and unsuitable for use as a balm (too flowable) based on visual thickness.

[0218] A deodorant stick formulation was made using Micrococcus luteus Q24 ingredient with a Dv90 of 290 μm according to the process described in WO 2006 / 104403: Example 2. Formulation 2 was highly viscous, with a uniform distribution of ingredients and a mostly solid consistency; it was initially solid and had no grittiness upon dissolution, and was not suitable for use as a balm (too viscous) based on visual viscosity.

[0219] Cetomacrogol cream formulations were made by blending gamma-irradiated Q24 raw materials (passed through a 250 μm sieve) with commercially available cetomacrogol cream (HealthE Non-ionic cream, Jaychem, New Zealand) in accordance with the Australia and New Zealand Clinical Trials Registry (ANZCTR) trial ACTRN12616000022460. Formulation 3 was moderately viscous, maintained uniform distribution of raw materials when dispensed, was not gritty, and was suitable for application to facial skin based on visual viscosity. Micrococcus luteus Q24 was found to be highly unstable in formulation 3. When stored at 25°C / 60% RH, a 7-log drop was observed after just 7 days, and no viable cells were found by day 14.

[0220] Comparative study - Microbiological stability The formulation was prepared by weighing 19.8 g of cetomacrogol 100BP cream (HealthE, Non-ionic cream, B62236, Jaychem, New Zealand) into a stomacher pouch using a sterile spoon. 0.2 g (2% of the final formulation) of Micrococcus luteus Q24 raw material was added to the cetomacrogol cream, sealed, and manually mixed until a homogeneous cream was formed. The cream was then placed in a stomacher and mixed for an additional 5 minutes. The cetomacrogol cream containing Micrococcus luteus Q24 was then dispensed into 30 ml glass vials and counted in triplicate. In a microcentrifuge tube (Eppendorf, USA), 0.1 g of Micrococcus luteus Q24 serum or cream was weighed and diluted with 0.9 g of warm (37°C) phosphate-buffered saline and polysorbate 80 (0.1% w / w). The mixture was homogenized using a vortex mixer (automatic vortex) by shaking at 2800 rpm for 5 minutes under ambient conditions (20°C ± 2°C) to obtain a homogenous dispersion. Then, 100 μL was serially diluted appropriately with PBS, spread onto hBaCa agar plates, and incubated at 37°C and 5% CO2 for 28–48 hours. Colonies were counted, and bacterial concentrations were calculated in CFU / g 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, and stability tests were performed at 7 and 14 days. Micrococcus luteus was found to be highly unstable in this formulation. When stored at 25°C / 60% RH, a 7-log drop was observed after just 7 days, and no viable cells were found by day 14.

[0221] Spreadability The spreadability of the balm formulations (Table 5) was measured using a procedure similar to that described by Saleh et al., "Evaluation of Skin Permeation and Analgesic Activity Effects of Carbopol Lornoxicam Topical Gels Containing Penetration Enhancer," The Scientific World Journal, (2014). A 1 cm diameter circle was drawn on a glass slide, and the balm was spread evenly within the 1 cm diameter. A second glass slide was placed over this, and then a 200 g weight was added, taking care to maintain a parallel downward angle. The weight was left on the slide for 30 seconds, then lifted, and the change in diameter was measured. The results are shown in Figure 4. [Table 6]

[0222] Spreadability properties are important for the application of balms to the skin. These results show that the order of balm spreadability is BLT-BB2 > BLT-BB9 > BLT-BB5 (Figure 4). BLT-BB2 had no beeswax content (only cetostearyl alcohol), while BLT-BB5 had a high proportion of beeswax.

[0223] Example 7: Physical Stability A centrifugal acceleration syneresis test was conducted to compare the syneresis tendency of balm formulations BLT-BB143, BLT-BB15, BLT-BB23, BLT-BB24, BLT-BB25, and BLT-BB26 with that of a deodorant stick formulation made according to the process described in Example 2 of WO 2006 / 104403 after 20 minutes of centrifugal acceleration at speeds of 1000, 1500, 5000, and 13,000 rpm. One gram of each balm formulation was weighed into a separate 1.5 mL Eppendorf tube and centrifuged at 13,000 rpm for 20 minutes. The syneresis fraction was transferred to another Eppendorf tube and weighed. This method was repeated for other centrifugation speeds (1500, 5000, and 13,000 rpm).

[0224] At 13,000 rpm, the glyceryl monostearate-based formulations (BLT-BB14 and BLT-BB23) showed low cumulative syneresis of only 11.6% and 5.8% after 20 minutes of centrifugal acceleration, while the beeswax and cetostearyl alcohol-based formulations BLT-BB24 and BLT-BB26 showed less physical stability with cumulative syneresis of 20% and 17.9% after 20 minutes of centrifugal acceleration (Figure 5A). This suggests that BLT-BB15 was the most physically stable formulation, with cumulative syneresis of only 4.5% after 20 minutes. All balm formulations tested showed less than 20% syneresis at 13,000 rpm. Therefore, the balms are likely to remain intact when exposed to "real-world" forces.

[0225] Unlike the balm formulation, the hard deodorant stick formulation of Example 2 of WO 2006 / 104403 did not exhibit any syneresis up to 5,000 rpm. At 13,000 rpm, it showed a higher cumulative syneresis of about 19%.

[0226] Unlike the balm formulations, all of the serum formulations shown in Table 4 showed greater than 20% syneresis at 5,000 rpm (Figure 5B). At 13,000 rpm, the serum formulations showed higher cumulative syneresis of 30-60%.

[0227] Interestingly, although formulations BLT-BB15 and BLT-BB24 differ only in the use of an oily base, BLT-BB15, which contains olive oil, is more stable with respect to syneresis than formulation BLT-BB24, which contains MCT oil.

[0228] Figure 5A shows syneresis measurements for slower centrifugation speeds of 1000, 1500 and 5000 rpm. As can be seen, syneresis increases with increasing centrifugation speed (rpm) for all formulations at 20 minutes. It is also clear that less syneresis occurs at the lower speeds (1000 and 1500 rpm). This supports the conclusion that balm formulations are likely to have good physical stability when exposed to "real-world" shear forces.

[0229] Example 8: Safety and Efficacy - EpiDerm Model The safety and efficacy of Micrococcus luteus Q24 bacterial cells was evaluated using an EpiDerm 3D skin model.

[0230] methodology Cultivation of EpiDerm Human 3D Skin Model (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 prewarmed differentiation medium (EPI-201-DM). They were then maintained at 37°C in a 5% CO 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.

[0231] Dose-response studies for tolerability Maturation of 24 immature EpiDerm tissues was performed as described above. Fully differentiated tissues were treated with either PBS (negative control) or Micrococcus luteus Q24 at five dose levels (1 x 10 5 cfu / ml ~ 1 × 10 9The tissues were randomly assigned to receive one of the following treatments (cfu / ml), with four replicate tissues used per treatment. The apical surface of the tissue was treated with 30 μL of treatment solution, and the tissues were then cultured at 37°C in a 5% CO atmosphere for 24 hours. At the end of that period, the tissues were gently rinsed 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.).

[0232] Safety and efficacy studies Maturation of 36 immature EpiDerm tissues was performed as described above. Fully differentiated tissues were numbered 1 through 36 and randomly assigned to receive one of six combinations of one of three main treatments (PBS, Micrococcus luteus Q24, or vitamin C) plus either PBS or 0.5% sodium dodecyl sulfate (SDS) exposure treatment (+0.5% SDS) as a negative control, and randomly placed in each of five 6-well cell culture plates. The remaining six tissues were to undergo histological examination or otherwise receive 5% SDS and were grouped together on 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. Starting on day 0, each tissue was treated daily with the appropriate main treatment solution (PBS, Micrococcus luteus Q24, or vitamin C) for 4 days, after which the tissue was returned to an incubator maintained at 37°C in a 5% CO2 atmosphere. For tissues treated with Micrococcus luteus Q24 or PBS vehicle, 30 μL of the appropriate solution was applied to the apical surface of the tissue after rinsing with PBS to remove any residue from the previous day's dose. Meanwhile, vitamin C (50 μg / mL) was provided in the culture medium. On day 4, conditioned medium samples were collected and stored frozen at -80°C for cytokine measurements. Thirty-three tissues were exposed to 30 μL of PBS containing 0.5% or 5% SDS, or PBS as a negative control. After 1 hour, the tissues were rinsed with PBS, patted dry with a cotton swab, and then the appropriate main treatment (PBS, Micrococcus luteus Q24, or vitamin C) was applied again and transferred to plates containing fresh medium. The remaining three tissues (for histological examination) were simply fed with fresh medium and given the appropriate treatment. All tissues were returned to the incubator and placed at 37°C in a 5% CO2 atmosphere for an additional 24 hours. Day 5 conditioned medium was then collected and frozen at -80°C. All tissues were gently rinsed with PBS, and three tissues for histological examination were fixed in neutral buffered formalin. The viability of the other 33 tissues was assessed using the MTT viability assay.

[0233] MTT viability assay (safety) The EpiDerm tissues on the cell culture inserts were added to 6-well cell culture plates containing 0.9 mL of DMEM medium (Thermo Fisher Scientific) containing 1 mg / mL MTT and incubated at 37°C under a 5% CO2 atmosphere for 3 hours. They were then rinsed with PBS and patted dry with cotton gauze. A cotton swab was used to remove any remaining moisture inside the inserts. Each insert and tissue was then immersed in 6 mL of propan-2-ol and allowed to soak overnight in a sealed 20 mL scintillation vial in the dark. The resulting purple solution of formazan dye was transferred to a disposable cuvette, and the absorbance was measured at 570 nm and 650 nm (reference wavelength) using an Ultrospec UV-Visible Spectrophotometer (LKB). Data analysis involved subtracting the absorbance at the reference wavelength from the 570 nm reading to obtain corrected absorbance values. These were converted to percent viability values ​​by dividing each absorbance value by the mean of the negative control group (PBS not exposed to 0.5% SDS) and multiplying by 100.

[0234] Histological examination (effectiveness) Tissues were fixed in neutral buffered formalin (LabServ) and sent to Gribbles Veterinary Lab (Christchurch, NZ) for preparation of H&E stained sections. Light microscopy was performed on a Leica DM6000 B microscope (Leica Microsystems, Switzerland), and images were acquired using Leica Application Suite v4.12 software.

[0235] Cytokine quantification for anti-inflammatory effects (efficacy) For the 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, a bead-based multiplex LEGENDplex™ assay (LEGENDplex™ Human Inflammation Panel 1 (13-plex); BioLegend, San Diego, CA, USA) was used according to the manufacturer's instructions. The relative activity observed for each of the measured cytokines is listed in Table 6. Reactions were performed in duplicate. Analysis was performed using a Cytek™ Aurora flow cytometer (Cytek Biosciences Inc., Fremont, CA, USA). Data were analyzed using Legendplex V8.0 software (BioLegend) and expressed in pg / mL. [Table 7]

[0236] 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), with multiple comparison correction using the Dunnett or Sidak method as appropriate.

[0237] Tolerability The tolerance of Micrococcus luteus Q24 was determined by a dose-response experiment. Live Micrococcus luteus Q24 (1.42 × 10) in phosphate-buffered saline (PBS) was used. 5 , 1.31×10 6 , 1.46×10 7 , 1.71×10 8 , and 1.63 × 10 9 EpiDerm tissue treated with 1 x 10 cfu / ml suspension showed >90% viability (Figure 6). 9Although the viability of CFU / ml was significantly different from the PBS control, the average cell (%) was still >90% viable, indicating that Micrococcus luteus Q24 was well tolerated by the model skin tissue during the 24-hour exposure.

[0238] These in vitro results closely resembled in vivo studies of Micrococcus luteus Q24 hydration serum, which showed a 1.5 × 10 8 No skin irritation was observed even after repeated administration of CFU / dose.

[0239] Conclusions: Micrococcus luteus Q24 at various clinically relevant doses was well tolerated in tissue culture models, demonstrating the safety of this strain and products containing Micrococcus luteus Q24.

[0240] safety After dose-response experiments, 1 x 10 8 The dose of CFU / ml was selected for safety and efficacy studies.

[0241] Measurements of 13 cytokines (days 0 and 4) were performed using Micrococcus luteus Q24 (1 × 10 8CFU / ml), PBS (negative control), and vitamin C (positive control). Of the 13 treatments, effects related to only three cytokines were observed: interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-18 (IL-18) are pro-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 Micrococcus luteus Q24 and PBS (Figure 7, upper left). IL-8 levels decreased slightly on day 4 for Micrococcus luteus Q24 and PBS (the change was significant only for PBS) and remained unchanged for vitamin C (Figure 7, upper right). In the case of IL-18, there was no significant difference between the levels on days 0 and 4, but Micrococcus luteus Q24 and vitamin C tended to show lower values ​​on day 4 (Figure 7, bottom center).

[0242] Conclusion: The results indicate that Micrococcus luteus Q24 is safe, as it did not induce an inflammatory immune response in epidermal tissues after daily administration for 4 days.

[0243] Effectiveness (organizational structure) 1×10 8A PBS suspension of live Micrococcus luteus Q24 at a dose of 100 CFU / ml, PBS (negative control), and vitamin C (positive control) were applied to EpiDerm cells for a 4-day period, and treatment-related changes in tissue morphology were recorded (n=1, Figure 8). While no obvious treatment-related differences were observed, tissue treated with Micrococcus luteus Q24 revealed trapped nuclear remnants in the stratum corneum, possibly indicating accelerated stratum corneum production. The stratum corneum was also slightly thicker in Micrococcus luteus Q24 tissue than in those treated with PBS or vitamin C. While more replicates are needed to confirm these findings, Micrococcus luteus Q24 appears to have a positive effect. The stratum corneum primarily functions as a barrier between the deeper layers of the skin and the external environment, preventing toxins and bacteria from entering the body. The stratum corneum also helps prevent water from evaporating into the atmosphere and is therefore important for maintaining skin hydration.

[0244] These results indicate that Micrococcus luteus Q24 may help rejuvenate and hydrate the skin by accelerating the formation of the stratum corneum.

[0245] Conclusion: Micrococcus luteus Q24 is effective in accelerating the growth of the stratum corneum, which is involved in barrier function and moisture retention. These in vitro results support the in vivo findings of increased moisture retention and reduced pores and wrinkles observed by study participants upon application of Micrococcus luteus Q24 Moisture Retention Serum.

[0246] Anti-inflammatory effects Only IL-6, IL-8, and IL-18 had detectable levels to determine the effects of treatment with Micrococcus luteus Q24, PBS (negative control), and vitamin C (positive control) (Figure 9 - top left).

[0247] Pretreatment with Micrococcus luteus Q24 caused a small, nonsignificant decrease in the amount of IL-6 released in response to 0.5% SDS; in contrast, vitamin C-treated tissues showed a slight increase on day 5 in response to 0.5% SDS exposure. A significant increase in IL-8 levels was evident on day 5 in response to 0.5% SDS for all three main treatment groups compared with day 4 (Figure 9, upper right). IL-18 release was also stimulated by 0.5% SDS in all groups, although the change was not significant for the Micrococcus luteus Q24 group (Figure 9, lower center).

[0248] Conclusion: The slight decrease in SDS-induced IL-6 release and the blunted increase in IL-18 for the Micrococcus luteus Q24 + 0.5% SDS group indicate the anti-inflammatory effect of Micrococcus luteus Q24.

[0249] Example 9: Sensory Test The sensory properties of balm formulations based on different platforms (beeswax, beeswax and cetostearyl alcohol, self-emulsifying glyceryl monostearate, and candelilla wax) were evaluated in a sensory test with healthy human volunteers. In this single-blind study, four lead formulations, BLT-BB9, BLT-BB15, BLT-BB14, and BLT-BB18, were randomly assigned letters A, B, C, or D for anonymity and given to participants. Participants evaluated the sensory and physical properties of the formulations using a questionnaire by applying the formulations to the back of their hand and were asked to score (1 to 5, where 1 = very poor and 5 = very good) the following parameters: ease of application, viscosity, spreadability, roughness, waxiness, oiliness, absorbency, moisture retention, skin feel after application, odor, and appearance. The rankings of these properties for the four different balms were then compared.

[0250] During the sensory testing, the four different balm formulations were ranked by the participants on various attributes. As shown in Figure 10, all four balm formulations (BB9, BB14, BB15, and BB18) were ranked 3 or higher, suggesting that all formulations were well accepted by the participants. This trend was consistent with the other attributes recorded (appearance and smell), which showed similar rankings.

[0251] To help distinguish between the balms, each sensory attribute was ranked and the total number of attributes identified as "highest ranked" for a given balm formulation was tabulated, as shown in Figure 11. For example, for "ease of application," BLT-BB15 and BLT-BB18 had an average rank of 4.7 out of 5, while BLT-BB9 and BLT-BB14 had average ranks of 4.1 and 3.75 out of 5, respectively. As a result, BLT-BB15 and BLT-BB18 were identified as the preferred formulations for this attribute.

[0252] Formulation BLT-BB15 ranked highest in overall sensory performance (score of 14). Although BLT-BB9 ranked highly (score of 10, FIG. 11), it was not carried forward for further evaluation; instead, the higher-ranked BLT-BB15, which has the same beeswax platform, was selected. The plant-based formulation BLT-BB14 was also selected for further evaluation in sensory and efficacy testing.

[0253] Example 10: Sensory and Efficacy Testing A single-blind, randomized, controlled, in-house study was conducted with 12 healthy subjects. The balms used in this study contained a 1% concentration of Micrococcus luteus Q24 (approximately 1E+9 cfu / g per application). Participants were provided with either Balm A (BLT-BB14) or Balm B (BLT-BB15), which they applied twice daily (morning and night) for 7 days to their left forearm (primary site) and another area of ​​concern (Table 7), chosen by the subjects as the location of dry / unhealthy skin (left elbow, left face-cheek, left leg, or left upper arm). [Table 8]

[0254] Skin parameters (moisture, keratinocytes, sebum, blemishes, wrinkles, pores, and impurities) were measured on days 0, 4, and 7 of the study and recorded using an advanced non-invasive skin analyzer (dpViso, Chowis, South Korea).

[0255] During testing, the same area was measured each time. However, this was more difficult in tricky locations such as the elbow. To minimize variability, participants placed their left hand on their left shoulder with their elbow elevated at a 90-degree angle. In addition to the skin analyzer, keratin parameters were tested using keratin strips. The strips were placed adhesive-side down on the area of ​​concern and light pressure was applied. They were left on for 10 seconds, then removed and analyzed using a skin analyzer (DermoPrime Viso, CHOWIS, South Korea) and DermoPrime software.

[0256] Comparison of the recorded data can be used to show improvements in skin parameters at specific sites compared to baseline and between different balm groups. Additionally, after the study participants completed a survey to evaluate the sensory properties of the formulations.

[0257] Statistical analysis was performed using MS Excel. A paired two-sample Student's t-test was used to compare participant data from different days and different formulations, with a significance level of p≦0.05. During statistical analysis, the significance level was expressed as follows: ns = not significant; p≦0.05 * p-value less than =0.05 p≦0.05 ** p-value less than =0.01 p≦0.05 *** p-value less than =0.001 p≦0.05 **** p-value less than =0.0001

[0258] In addition to investigating changes in skin parameters due to the balm formulations, the study also investigated the sensory properties of the balms, specifically how participants perceived long-term balm use.No statistically significant differences were observed between the ages of subjects who applied Balm A or Balm B (p ≤ 0.05).

[0259] There were a total of 12 participants in this study, designated by participant number (P1-P12), with varying participant characteristics (age and gender) (Table 7). The 12 participants were divided into two groups and assigned to use either Balm A or Balm B (Table 7). The mean ages of the two groups were 34.3 and 34 years, respectively. A one-way analysis of variance was used to compare the ages of subjects who applied Balm A and Balm B, yielding a p-value of 0.9, confirming that there was no statistical difference between the ages of the two groups.

[0260] Data from participant 6 (P6) was identified as an outlier and removed from the analysis. A similar approach was taken for data outliers related to impurities. Data outliers for other skin parameters were not removed because they were less common and did not tend to show significant differences relative to other raw data values.

[0261] Test results - Main area (left forearm) The percentage of participants showing changes in measured key site skin parameters is shown in Figure 12 for Balms A and B, respectively. Data is presented as two separate bars, "Day 4" and "Day 7." Day 4 describes the changes observed between days 0 and 4 of the study, and Day 7 describes the changes observed between days 0 and 7.

[0262] Results for Balm A show improvement in more participants for the skin parameters moisture, sebum and blemishes within the first four days of product application, while results for Balm B show improvement in more participants for all skin parameters over the full seven-day study.

[0263] These results indicate that Balm A showed a more rapid improvement in skin parameters. However, the number of participants who showed improvement tended to decrease by day 7 of the study. For example, 60% of participants showed increased moisture by day 4 of the study, but this decreased to 40% of participants by day 7 (Figure 12).

[0264] On the other hand, Balm B showed improvement in skin parameters in more subjects between days 0 and 7. For example, by day 4, 33% of participants showed improvement in moisture, which increased to 67% by day 7. This trend suggests that longer application of Balm B may result in improvement in skin parameters in more test participants. Furthermore, skin improvement was not diminished by day 7, which also suggests that Balm B may be able to maintain its improvement over a longer period of time. The skin parameters of pores, age spots, wrinkles, and impurities for both Balm A and Balm B improved only minimally over the test period, and the number of participants who showed improvement was small. This result is expected given the test period, as the forearm is a generally healthy area of ​​skin and is free of pore or wrinkle imperfections.

[0265] The data collected for each of the key area characteristics was compared to determine whether there were statistically significant differences between the skin parameter values ​​over time. As shown in Figure 12, improvements were observed for sebum, pores, blemishes, and wrinkles, with the most significant improvement occurring in the skin parameter of moisture. For Balm B, a statistically significant change was observed between days 0 and 7 of the study. Interestingly, the majority of statistically significant differences observed were in participants who applied Balm B, while Balm A showed fewer statistically significant changes between time points. The most frequently observed trend was that the intermediate time point (recorded on day 4) was lower or higher than those measured on days 0 and 7. Therefore, for the key areas, most skin parameters remained unchanged over the study period. This result is expected. The forearm is already a healthy area of ​​skin, and for many subjects, it is naturally more hydrated and less keratinized.

[0266] Test results - Areas of concern (varies by participant) The areas of concern were selected by the subjects as locations with dry or unhealthy skin, including the left elbow, left face-cheek, left leg, or left upper arm. In addition to testing the skin parameters of moisture, sebum, pores, blemishes, wrinkles, and impurities, keratin was also measured for these areas.

[0267] As shown in Figure 12, 100% of participants (both Balm A and Balm B) showed a decrease in the amount of callus (dry skin) in the areas of concern by day 7 of the study. A similar trend was observed for moisture, with 100% of participants (both Balm A and Balm B) showing an increase in moisture by day 7.

[0268] Additionally, an increase in sebum is observed with both Balms A and B. Sebum is associated with acne, but it has also been observed that it is generally directly proportional to moisture. As moisture increases in this system, so does sebum.

[0269] While some subjects showed improvement in the skin parameters of pores, blemishes, wrinkles, and impurities with both balms, Balm B showed a more pronounced improvement among a larger number of participants. With Balm B, over 50% of participants showed improvement in each of those skin parameters (excluding impurities), while less than 50% of participants using Balm A experienced improvement in those skin parameters.

[0270] In summary, it can be concluded that Balm B showed improvement in the skin of more subjects in the skin parameters of pores, blemishes, wrinkles and impurities, while both Balm A and Balm B showed clear improvement in hydration and exfoliation.

[0271] Statistical analysis - Areas of concern (moisture and keratin) While the trends observed by percentage of participants indicate that changes are occurring, comparison of the raw data is necessary to better predict whether the balm is providing statistically significant improvements to skin quality parameters.

[0272] Analysis of moisture skin parameters is shown in Figures 13 and 14. The moisture scores of the subjects who used Balm A showed no statistically significant difference between days 0 and 4 (P = 0.2), and a weakly significant difference between days 4 and 7 (p = 0.073). However, there was a statistically significant difference between the moisture scores on days 0 and 7 (p = 0.043). In other words, the significant increase in moisture in the participants who used Balm A was mainly observed after using the balm for 7 days. This indicates that Balm A has an excellent moisture-retaining effect. In addition, since the moisture scores increased over time, it can be expected that long-term use of the product will further improve moisture skin parameters.

[0273] In contrast to the results for Balm A, participants who used Balm B showed statistically significant differences in moisture scores for the areas of concern by Day 4. This indicates that Balm B provided an immediate moisture improvement that was maintained throughout the 7-day study. Similarly, moisture scores for Balm B further improved by Day 7. Therefore, continued use of the product can be expected to further improve moisture skin parameters.

[0274] The consistent and statistically significant increase in moisture over time experienced by subjects using Balm B suggests that it was more successful at improving moisture over time than Balm A. This is supported by the higher mean moisture scores at Day 7 for participants using Balm B (30 and 38.5 for Balm A and Balm B, respectively).

[0275] The change in the scores for the keratin skin parameters for Balms A and B is shown in Figures 13 and 14. As can be seen, there are statistically significant differences between each time point. For Balm A, there was a decrease between days 0 and 4 and a slight increase between days 4 and 7, indicating that Balm A showed an immediate decrease in dry skin after use, but that keratin again increased slightly after one week of regular application. Importantly, the keratin levels on both days 4 and 7 were lower than on day 0, thus indicating continued improvement by the end of the study.

[0276] The callus score for Balm B showed an initial large decrease (days 0-4, p=≦0.05), similar to that observed for Balm A. The lower callus score was maintained through day 7.

[0277] Statistical analysis - areas of concern (other skin parameters) In addition to moisture and keratin, other skin parameters (wrinkles, impurities, blemishes and pores) were also investigated and are graphed in Figures 13 and 14 for Balm A and Balm B, respectively.

[0278] Various statistically significant differences occur between the skin parameters wrinkles, impurities, age spots, and pores for both Balm B and Balm A in the areas of concern. However, these statistically significant relationships are variable and do not appear to indicate a strong trend for improvement over time. Interestingly, Balm B showed a statistically significant reduction in wrinkles.

[0279] In addition to comparing Balm A and Balm B in the primary area (forearms) and areas of concern, the results for moisture and callus scores were also analyzed by participant age. Participants in the study were categorized by age into younger Group A (21-32 years) and older Group B (38-50 years), providing two groups of similar size (Table 8).

[0280] There was a statistically significant age difference between these two groups, with a mean age of 24.8 and 44.2 years, respectively, with a p-value of 5.47E-6. [Table 9] * Exclude from analysis

[0281] The skin parameter data for moisture and keratin for these two groups were compared to determine whether the younger group (Group A) or older group (Group B) showed statistically significant differences over the 7-day study period. The mean data and p-values ​​for these comparisons are provided in Figure 15.

[0282] As shown in Figure 15, both Group A (young) and Group B (old) demonstrate an increase in hydration scores over time. Young Group A demonstrates mean hydration scores of 10.2, 16.0, and 29.8 on days 0, 4, and 7, respectively, while Older Group B demonstrates mean hydration scores of 10.0, 15.2, and 38.7 on days 0, 4, and 7, respectively. While both groups demonstrate an increase in hydration, Group B's hydration scores demonstrate a more statistically significant difference over time, with significant p-values ​​(p=0.005 and p=0.014) from days 0 to 7 and from days 4 to 7. Group A did not demonstrate statistically significant differences among the three time points for hydration scores. Thus, the older participants (Group B) demonstrated a more significant increase in hydration over the study period.

[0283] Group A shows a gradual decrease in callus scores over time (mean scores of 54.6, 19.9, and 14.9 on days 0, 4, and 7, respectively), while Group B shows a fluctuating trend (Figure 15). This trend shows a decrease in callus between days 0 and 4 (mean callus scores of 35.9 and 15.9, respectively), which increases to 26.3 by day 7. Importantly, while a slight increase is observed between days 4 and 7, the callus value on day 7 is still lower than the baseline callus measurement. Thus, callus is still observed to decrease over time during balm application.

[0284] Both Group A and Group B showed statistically significant differences in calluses across all time points, meaning that a significant decrease in calluses was observed throughout the study for both Group A and Group B.

[0285] Example 11: Addition of prebiotics Inhibition of common prebiotics CABK12 agar plates were divided into six segments and lawned with a suspension of Micrococcus luteus Q24 feedstock. Each aqueous prebiotic candidate to be screened was serially diluted in sterile distilled water at concentrations ranging from 100% to 0.3%. Oil-based substances were tested at 100% only.

[0286] 20 μL of each substance at each concentration was pipetted onto a spot on one segment of a confluent CABK12 agar plate and incubated at 37° C. in 5% CO 2 in air for 24 hours. [Table 10]

[0287] Conclusions: All substances passed the initial screening test for products without M. luteus Q24, except for green tea powder, which was inhibitory at all concentrations tested. Six other prebiotic candidates showed some inhibitory effect on M. luteus Q24, indicating that they may not be suitable for use in products requiring viable M. luteus Q24 stability, but may have potential for use in prebiotic-only formulations.

[0288] Effect of prebiotics on the growth of Micrococcus luteus Q24 A batch of M17 broth (Difco #218561) was made according to the manufacturer's instructions, except for the lactose solution. 50 ml of broth was dispensed into sterile 100 ml Schott bottles, and 2.5 g (5% w / v) of each prebiotic candidate was added and mixed thoroughly using a magnetic stirrer and stir plate. The mixture was autoclaved at 110°C for 10 minutes and allowed to cool.

[0289] Suspensions of prebiotic candidates were preheated to 40°C to enhance homogenization of any oil-based components before dispensing into wells. 2 ml of each suspension and M17 alone (control) were pipetted into sterile 24-well tissue culture plates. A suspension of Micrococcus luteus Q24 source component was made in PBS and adjusted to an optical density of 0.125. 100 μl of suspension was pipetted into each well. Upon addition of the suspension, each well was mixed by aspirating and dispensing the solution five times with a 1 ml pipette.

[0290] Each sample was counted at 0, 3, 6, 15, 26, and 34 hours using the following method. At each time point, the wells were mixed using a 1 ml pipette, dispensing and aspirating five times. 100 μl was then removed from each well and added to an Eppendorf tube containing 900 μl of 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 to 900 μl of fresh PBS, and this process was repeated until six serial dilutions were achieved. 20 μl of each dilution from each sample was spotted in triplicate onto sheep blood agar plates. The spots were allowed to dry for 30 minutes and then placed in a 37°C, 5% CO2 incubator for 28–36 hours. The number of colonies in each spot was then counted using an electronic colony counter and averaged to obtain the final results.

[0291] Results: All prebiotic candidates tested showed a higher increase in Micrococcus luteus Q24 than the control, with most resulting in a 1-2 log increase over 24 hours compared to the control.

[0292] CONCLUSIONS: Surprisingly, commonly used prebiotics typical for topical application (carbohydrates (except oatmeal)) did not support the growth of Micrococcus luteus Q24.

[0293] Traditional substances used as prebiotics for gut bacteria did not perform well in increasing the growth of Micrococcus luteus Q24, and some, such as xylitol and manuka honey, had a negative effect on the number of viable Micrococcus luteus Q24 cells at 24 hours (Figure 16).

[0294] In contrast, oil-based substances typically used as functional actives and other cosmetic ingredients performed very well, with the best increasing viable cell counts of Micrococcus luteus Q24 by 2 logs over 24 hours.

[0295] Prebiotic Combinations The same methodology as above was used, except that the time points tested were 0, 6, 18, and 24 hours. Olive squalene was selected as one of the preferred prebiotics, and the other three were analyzed in combination with olive squalene and compared to the results of the prebiotic candidates alone (Figure 17).

[0296] Results: The growth of Micrococcus luteus Q24 increased more than the sum of olive squalene and pomegranate seed oil alone, and the combined use of both prebiotics showed a synergistic response leading to an improved growth rate of Micrococcus luteus Q24.

[0297] Growth of Micrococcus luteus Q24 was equivalent to the sum of olive squalene and oatmeal flour (colloidal oatmeal) alone, indicating no synergistic response with this combination.

[0298] The growth of Micrococcus luteus Q24 increased more than the sum of olive squalene and vitamin E alone, and the use of both prebiotics together showed a synergistic response resulting in an improved growth rate of Micrococcus luteus Q24.

[0299] Conclusions: Both olive squalene + pomegranate seed oil and olive squalene + vitamin E were found to have a synergistic relationship in improving the growth rate of Micrococcus luteus Q24 compared to either alone. Olive squalene and oatmeal flour did not show this same response. Formulations made to contain both olive squalene and either pomegranate seed oil or vitamin E were more effective than formulations containing only olive squalene, pomegranate seed oil, or vitamin E. JPEG2025529203000011.jpg220160

Claims

1. A topical composition comprising Micrococcus luteus Q24 and a viscosity adjusting agent, the topical composition having a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C.

2. Micrococcus luteus Q24 was approximately 1 x 10 ３ ~Approx. 1×10 １２ 10. The topical composition of claim 1, comprising:

3. 3. The topical composition of claim 2, comprising one or more viscosity modifiers in a total amount of about 30 to about 80% w / w.

4. 4. The topical composition of claim 2 or 3, wherein the viscosity modifier is selected from the group consisting of hydrophobic silica, hydrophilic silica, white wax, yellow wax, ethyl cellulose, emulsifying wax, cocoa butter, shea butter, fatty alcohols, glyceryl monostearate, candelilla wax, lanolin, and combinations of any two or more thereof.

5. 5. The topical composition of claim 4, wherein the viscosity modifier is selected from a combination of white wax or yellow wax and cetostearyl alcohol.

6. The topical composition of any one of claims 1 to 5, comprising a dispersing agent.

7. 7. The topical composition of claim 6, comprising the dispersant in an amount of about 0.1 to about 5% w / w.

8. 8. The topical composition of claim 6 or 7, wherein the dispersing agent is selected from the group consisting of polysorbate 80, polysorbate 20, sorbitan oleate, egg lecithin, soy lecithin, polyoxyl 35 castor oil, sodium stearoyl glutamate, sodium cocoyl isethionate, cetearyl olivate, and combinations of any two or more thereof.

9. The topical composition of any one of claims 1 to 8, further comprising an oily base.

10. 10. The topical composition of claim 9, wherein the oily base is selected from the group consisting of medium chain triglyceride (MCT) oil, vegetable oil, or a combination thereof.

11. 11. The topical composition of claim 10, wherein the MCT oil is caprylic / capric triglyceride oil.

12. 11. The topical composition of claim 10, wherein the vegetable oil is selected from the group consisting of sunflower oil, canola oil, soybean oil, olive oil, jojoba oil, argan oil, rosehip oil, marula oil, chamomile oil, tamanu oil, grapeseed oil, calendula oil, pomegranate oil, macadamia oil, barberry fruit oil, sweet almond oil, evening primrose oil, and combinations of any two or more thereof.

13. 11. The topical composition of claim 10, wherein the vegetable oil is selected from the group consisting of MCT oil, olive oil, calendula oil, pomegranate oil, and combinations of any two or more thereof.

14. 1. A topical composition comprising: about 0.5-2% w / w of Micrococcus luteus Q24; about 15 to about 35% w / w beeswax; about 2 to about 10% w / w cetostearyl alcohol; about 10% to about 30% w / w cocoa butter; A sufficient amount of oily base, Including, A topical composition, wherein the composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C.

15. 1. A topical composition comprising: Approximately 1×10 ３ ~Approx. 1×10 １２ cfu / g of Micrococcus luteus Q24; about 15 to about 35% w / w beeswax; about 2 to about 10% w / w cetostearyl alcohol; about 10% to about 30% w / w cocoa butter; A sufficient amount of oily base, Including, A topical composition, wherein the composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C.

16. 1. A topical composition comprising: about 0.5-2% w / w of Micrococcus luteus Q24; about 10 to about 30% w / w beeswax; about 10% to about 30% w / w shea butter; about 10% to about 30% w / w cocoa butter; A sufficient amount of oily base, Including, A topical composition, wherein the composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C.

17. 1. A topical composition comprising: Approximately 1×10 ３ ~Approx. 1×10 １２ cfu / g of Micrococcus luteus Q24; about 10 to about 30% w / w beeswax; about 10% to about 30% w / w shea butter; about 10% to about 30% w / w cocoa butter; A sufficient amount of oily base, Including, A topical composition, wherein the composition has a viscosity of about 2,500,000 to about 8,000,000 cP at 25°C.

18. 18. The topical composition of any one of claims 14 to 17, further comprising about 0.5 to about 2% w / w of polysorbate 80.

19. 19. The topical composition of any one of claims 14 to 18, wherein the oily base is a medium chain triglyceride oil or olive oil.

20. 20. The topical composition of any one of claims 1 to 19, further comprising 0.1 to 35% w / w of prebiotic(s).

21. 21. The topical composition of claim 20, wherein the prebiotic(s) are selected from olive squalene, pomegranate seed oil, vitamin E, or combinations thereof.

22. 22. The topical composition of claim 20 or 21, wherein the prebiotic(s) are selected from a combination of olive squalene and pomegranate seed oil; a combination of pomegranate seed oil and oatmeal; a combination of olive squalene and vitamin E; and a combination of olive squalene, pomegranate seed oil and vitamin E.

23. 23. The topical composition of any one of claims 1 to 22, wherein the Micrococcus luteus Q24 is free of lyoprotectants.

24. The topical composition of any one of claims 1 to 23, which is preservative-free.

25. 25. The topical composition of any one of claims 1 to 24, further comprising one or more additional probiotic(s).

26. 26. The topical composition of claim 25, wherein the one or more additional probiotic(s) is selected from the group consisting of Streptococcus spp., Lactobacillus spp., Limosilactobacillus spp., Lacticaseibacillus spp., Ligilactobacillus spp., Lactiplantibacillus spp., Bifidobacterium spp., Saccharomyces spp., and combinations of any two or more thereof.

27. 27. The topical composition of claim 26, wherein the Streptococcus species is selected from the group consisting of Streptococcus salivarius K12, Streptococcus salivarius M18, Streptococcus 24SMB, Streptococcus oralis (e.g., S. oralis 89a), Streptococcus salivarius DB-B5, and combinations of any two or more thereof.

28. Each additional probiotic was approximately 1 x 10 ３ ~Approx. 1×10 １２ 28. The topical composition of any one of claims 25 to 27, comprising in an amount of 0.01 cfu / g.

29. 29. The topical composition of any one of claims 1 to 28, further comprising one or more postbiotic(s).

30. 30. The topical composition of claim 29, wherein the postbiotic is selected from the group consisting of Streptococcus ferment, Streptococcus ferment filtrate, Streptococcus ferment lysate, Streptococcus lysate, Streptococcus filtrate, Lactobacillus (including Rimosilactobacillus spp., Ligilactobacillus spp., and Lactiprancylbacillus spp.) ferment, Lactobacillus (including Rimosilactobacillus spp., Ligilactobacillus spp., and Lactiprancylbacillus spp.) filtrate, Micrococcus ferment lysate, Bifidobacterium ferment lysate, Bifidobacterium ferment filtrate, Galactomyces ferment filtrate, Saccharomyces ferment filtrate, Bacillus ferment, Bacillus filtrate, and combinations of any two or more thereof.

31. 31. The topical composition of any one of claims 1 to 30, further comprising 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, an inorganic pigment, an odor absorber or neutralizer, a sunscreen, and combinations of any two or more thereof.

32. 32. The topical composition of claim 31, wherein 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.

33. 32. The topical composition of claim 31, wherein 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.

34. 32. The topical composition of claim 31, wherein 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, salivaricin D, salivaricin M, salivaricin G32, salivaricin A5, and combinations thereof.

35. 32. The topical composition of claim 31, wherein the prebiotic is selected from the group consisting of manuka honey powder, olive squalene, 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, inulin, borago officinalis (borage) seed oil, pomegranate extract, luohan fruit extract, xylitol, yeast extract, fructooligosaccharide powder, fructooligosaccharide liquid, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, niacinamide, sunscreen, and combinations of any two or more thereof.

36. 32. The topical composition of claim 31, 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 combinations of any two or more thereof.

37. 32. The topical composition of claim 31, wherein the antioxidant is selected from the group consisting of vitamin E, resveratrol, squalene, vitamin C, beta-carotene, retinyl acetate, retinyl palmitate, retinol, niacinamide, caffeine, bakuchiol, licorice root, and combinations of any two or more thereof.

38. 32. The topical composition of claim 31, wherein 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, palmitic acid heptapeptide-15, 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, sunscreen, lactic acid, camellia oil, hemp seed oil, seabuckthorn fruit oil, lanolin, and combinations of any two or more thereof.

39. 32. The topical composition of claim 31, wherein the antimicrobial agent is selected from the group consisting of zinc, azelaic acid, benzoyl peroxide, tea tree oil, salicylic acid, and combinations thereof.

40. 40. The topical composition of any one of claims 1 to 39, further comprising about 0.1% to about 10% w / w olive squalene or about 0.1 to about 10% w / w pomegranate seed oil.

41. 41. The topical composition of any one of claims 1 to 40, further comprising about 0.1% to about 10% w / w olive squalene and about 0.1 to about 3% w / w vitamin E.

42. 42. The topical composition of any one of claims 1 to 41, which is non-aqueous.

43. 43. The topical composition of any one of claims 1 to 42, wherein the particle size (Dv90) of Micrococcus luteus Q24 is less than about 300 μm.

44. 43. The topical composition of any one of claims 1 to 42, wherein the particle size of the Micrococcus luteus Q24 is less than about 250 μm or less than about 100 μm.

45. 45. The topical composition of any one of claims 1 to 44, having a shelf life of at least 3 months at 25°C and 60% RH.

46. 46. ​​A method of improving the appearance of skin or at least one sign of aging comprising applying to the skin a topical composition according to any one of claims 1 to 45.

47. 47. The method of claim 46, wherein improving the appearance of skin or at least one sign of aging comprises skin looking more radiant, skin looking healthier, skin feeling more hydrated, pores being smaller, skin feeling softer, skin looking clearer, reduced wrinkles, reduced dryness, reduced age spots, reduced impurities, increased moisture, reduced exfoliation, and regulated sebum production.

48. 48. The method of claim 46 or 47, wherein improving the appearance of skin or at least one sign of aging comprises reducing dryness of the skin.

49. 49. The method of any one of claims 46 to 48, wherein improving the appearance of the skin comprises improving at least one of teething rash, diaper rash, or dry skin in an infant.

50. 46. ​​A topical composition according to any one of claims 1 to 45 for improving the appearance of skin or at least one sign of aging or for reducing dryness of the skin.

51. 46. ​​Use of a topical composition according to any one of claims 1 to 45 in the manufacture of a medicament for improving the appearance of skin or at least one sign of aging, or for reducing dryness of the skin.

52. 1. A method for preparing a topical composition comprising Micrococcus luteus Q24, comprising: a) heating a mixture of an oily base and a viscosity modifier to provide a molten mixture; b) cooling the molten mixture to a temperature in the range of about 35°C to about 28°C; c) adding Micrococcus luteus Q24 to the molten mixture from step b) to provide a homogenous mixture; d) cooling the mixture from step c) to room temperature to provide the composition. A manufacturing method comprising:

53. 53. The method of claim 52, wherein the molten mixture is cooled to about 30°C in step (b).

54. 1. A method for preparing a topical composition comprising Micrococcus luteus Q24, comprising: e) heating the mixture of oily base and viscosity modifier to provide a molten mixture; f) cooling the molten mixture to a temperature of about 22°C to about 27°C; g) adding Micrococcus luteus Q24 to the cooled mixture from step b) and mixing to provide a homogenous mixture. A manufacturing method comprising:

55. 55. The method of claim 54, wherein the molten mixture is cooled to room temperature in step (b).

56. 56. The method of any one of claims 52 to 55, further comprising adding a dispersant during step (a) or before step (b).

57. 57. The method of any one of claims 52 to 56, wherein the topical composition produced is as defined in any one of claims 1 to 45.

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