Probiotic skin preparations

Abstract

Described herein are dermatological compositions containing formulations of wild-type or transgenic keratinocyte bacteria and methods thereof for use on skin. The cosmetic and therapeutic formulations described herein can be used as a daily skin cosmetic, or for treating skin inflammation or disease, including acne, psoriasis, rosacea, dermatitis, or eczema.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 042,684, filed June 23, 2020. The above application is incorporated herein by reference. Technical Field

[0003] This article describes dermatological compositions containing wild-type or transgenic Cutibacterium bacteria and methods of application to the skin. The cosmetic and therapeutic formulations described herein can be used for daily skin care or to treat skin inflammation or conditions, including acne, psoriasis, rosacea, dermatitis, or eczema. Background Technology

[0004] The microbiome is a complex network of microorganisms that live in and on the human body. On the skin, there are billions of microorganisms, including bacteria, viruses, fungi, and archaea. These microorganisms can be commensal (beneficial), pathogenic (harmful), or symbiotic. A symbiotic relationship occurs when the skin's microbes depend on the human host, just as the host depends on the microbes. When this relationship becomes unbalanced, it can lead to problems such as opportunistic infections, inflammatory diseases, or accelerated aging. This is known as dysbiosis.

[0005] In particular, human skin contains a variety of symbiotic microorganisms essential for healthy skin. These symbiotic or commensal bacteria live in the upper regions of the epidermis and hair follicles. These microorganisms protect against transient pathogenic organisms by competing for nutrients, producing antimicrobial compounds, or stimulating the skin's immune system. While most skin flora are symbiotic, some specific bacteria can cause non-infectious skin conditions such as acne, psoriasis, rosacea, dermatitis, or eczema. For example, acne vulgaris is associated with high populations of certain *Cutibacterium acne*. This leads to excessive sebum production and skin inflammation common in acne. Typical treatment involves topical and systemic antibiotics, which kill not only the *Cutibacterium acne* but also other symbiotic bacteria.

[0006] Traditionally, skincare routines have focused heavily on the interaction between ingredients and human skin cells. However, ingredients such as antibacterial agents and irritating preservatives can inadvertently disrupt the skin's microbiome. Current research indicates that the microbes living inside and on the skin secrete beneficial substances that directly influence and are crucial to skin health. Therefore, promoting the growth of symbiotic microbes on the skin is essential.

[0007] Skincare products containing irritating ingredients can disrupt the microbiome, leading to dysbiosis, which includes an imbalance in the number of "beneficial" and "harmful" microorganisms (and the molecules they secrete).

[0008] What is needed now are dermatological compositions containing probiotic ingredients and dormant genetically modified organisms that can mitigate the harmful effects of pathogens while enhancing the skin's natural microbiome. Summary of the Invention

[0009] One embodiment described herein is an anhydrous dermatological composition comprising: about 0.1% to about 5% (by weight) of one or more wild-type or growth-arrested transgenic bacteria or probiotic components; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable anhydrous excipients. In one aspect, the dermatologically acceptable anhydrous excipients include one or more emollients, humectants, moisturizers, vitamins, vitamin analogs, gelling agents, rheology modifiers, pH adjusters, or preservatives; and optionally one or more solvents.

[0010] Another embodiment described herein is an anhydrous dermatological composition comprising: one or more growth-arrested transgenic microorganisms; and one or more dermatologically acceptable anhydrous excipients, including one or more emollients, humectants, vitamins, vitamin analogs, or gelling agents. In one aspect, this anhydrous dermatological composition maintains the growth-arrested transgenic microorganisms in a dormant state for at least 6 months and up to about 2 years.

[0011] Another embodiment described herein is an anhydrous dermatological composition comprising: about 0.1% to about 5% (by weight) of growth-arrested *C. acne* strains; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable anhydrous excipients, including one or more emollients, humectants, vitamins, vitamin analogs, or gelling agents. In one aspect, the anhydrous dermatological composition maintains the growth-arrested *C. acne* strains in a dormant state for at least 6 months and up to about 2 years. In another aspect, the composition comprises: about 1% to 2% (by weight) of growth-arrested *C. acne* strains; about 90% (by weight) of one or more emollients; and about 10% (by weight) of one or more gelling agents. In yet another aspect, the growth-arrested *C. acne* strains comprise lyophilized powder comprising: *C. acne*, sodium chloride, calcium stearate, and inulin (fructopolysaccharide). On the other hand, the emollients include ethyl olivate, olive (Olea europaea) leaf extract, and castor oil, and the gelling agent includes polyamide-3 (dilinoleic acid, ethylenediamine, PEG / PPG-32 / 10 aminopropyl methyl ether-terminated polypropylene glycol diamine). On another hand, the composition comprises: about 0.1-5% (by weight) of growth-arrested acne-causing keratinocytes; about 55-95% (by weight) of ethyl olivate and olive leaf extract; about 20-40% (by weight) of castor oil; and about 1-15% (by weight) of polyamide-3 (dilinoleic acid, ethylenediamine, PEG / PPG-32 / 10 aminopropyl methyl ether-terminated polypropylene glycol diamine). In another aspect, the composition comprises: about 1% to 2% (by weight) of growth-arrested acne-causing keratinocytes; about 1% (by weight) of vitamin A or a vitamin A analogue; about 90% (by weight) of one or more emollients; and about 10% (by weight) of one or more gelling agents. In another aspect, the composition comprises: about 0.1-5% (by weight) of growth-arrested acne-causing keratinocytes; about 0.1-5% (by weight) of retinol; about 0.1-5% (by weight) of vitamin A palmitate; about 50-75% (by weight) of ethyl olive oil and olive leaf extract; about 20-40% (by weight) of castor oil; and about 1-15% (by weight) of polyamide-3 (dilinoleic acid, ethylenediamine, PEG / PPG-32 / 10 aminopropyl methyl ether-terminated polypropylene glycol diamine).

[0012] Another embodiment described herein is an aqueous dermatological composition comprising: about 0.1% to about 5% (by weight) of a probiotic composition; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable excipients, including one or more humectants, moisturizers, rheology modifiers, pH adjusters, preservatives, or solvents. In one aspect, the probiotic composition comprises *Propionibacterium acnes* fermentation product, including a lyophilized powder of purified *Propionibacterium acnes* RoxP fermentation medium. In another aspect, the *Propionibacterium acnes* fermentation product is purified by filtration and percolation. In another aspect, the composition comprises: about 0.1% to 5% (by weight) of *Propionibacterium acnes* fermentation product; about 80% (by weight) of one or more solvents; about 10% (by weight) of one or more wetting agents; about 1% (by weight) of one or more moisturizing agents; about 2% (by weight) of one or more rheology modifiers; about 4% (by weight) of one or more film-forming agents; about 2% (by weight) of one or more preservatives; and about 1% (by weight) of one or more pH adjusters. On the other hand, the composition comprises: about 0.1-5% (by weight) of *Propionibacterium acnes* fermentation product; about 70-90% (by weight) of water; about 0.1-5% (by weight) of sodium hyaluronate solution; about 5-15% (by weight) of acetamide ethoxyethanol; about 1-5% (by weight) of polyurethane; and about 0.1-5% (by weight) of a solution of sodium protocatechuate, sodium lactate, protocatechuic acid, arginine, aspartic acid, glycine, alanine, serine, valine, proline, threonine, isoleucine, histidine, and phenylalanine. The composition comprises about 0.1-5% (by weight) of a copolymer of hydroxyethyl acrylate and sodium acryloyldimethyl taurate; about 0.1-5% (by weight) of polyacrylate crosspolymer-6; about 0.1-5% (by weight) of benzyl alcohol; about 0.5-1.5% (by weight) of sodium benzoate and potassium sorbate; and about 0.1-5% (by weight) of a citric acid solution (50%). In another aspect, the composition further comprises about 0.1% to about 2% (by weight) of retinoic acid.

[0013] Another embodiment described herein is a method for preparing a probiotic fermentation product, the method comprising: culturing microorganisms in a fermentation medium; separating the fermentation medium from the microorganisms; tangentially filtering the fermentation medium by exchange with a buffer to remove small molecule contaminants, thereby producing a purified fermentation product; and freeze-drying the purified fermentation product. In one aspect, the microorganism is *Propionibacterium acnes* RoxP (C. acnes RoxP).

[0014] Another embodiment described herein is a fermentation product produced by any of the methods or means described herein.

[0015] Another embodiment described herein is a method for enhancing the skin microbiome, comprising applying an effective amount of a dermatological composition comprising: about 0.1% to about 5% (by weight) of one or more growth-arrested genetically modified bacteria or probiotic components; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable excipients.

[0016] Another embodiment described herein is a method for minimizing the growth of pathogenic bacteria on the skin, the method comprising: applying an effective amount of a dermatological composition to the skin, the composition comprising: about 0.1% to about 5% (by weight) of one or more growth-retarded genetically modified bacteria or probiotic components; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable excipients.

[0017] Another embodiment described herein is a method for treating acne, comprising applying an effective amount of a dermatological composition to the skin, the composition comprising: about 0.1% to about 5% (by weight) of one or more growth-arrested genetically modified bacteria or probiotic components; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable excipients.

[0018] Another embodiment described herein is a method of preparing a cosmetic or therapeutic composition comprising: about 0.1% to about 5% (by weight) of one or more growth-arrested genetically modified bacteria or probiotic components; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable excipients.

[0019] Another embodiment described herein is a cosmetic or therapeutic composition prepared by any of the methods or means described herein.

[0020] Another embodiment described herein is a method of preparing a cosmetic or therapeutic composition comprising: about 0.1% to about 5% (by weight) of one or more growth-arrested genetically modified bacteria or probiotic components; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable excipients.

[0021] Another embodiment described herein is the use of a cosmetic or therapeutic composition comprising one or more growth-arrested genetically modified bacteria or probiotic ingredients; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable excipients for the treatment of skin diseases or ailments. Attached Figure Description

[0022] Figure 1The growth of Xycrobe cells (growth-arrested modified Propionibacterium acnes RT6) is shown to be dependent on the presence of L-(+)-arabinose. Xycrobe cells do not grow in media without L-(+)-arabinose, but begin to grow when arabinose is added.

[0023] Figure 2 shows that Xycrobe cells (growth-arrested modified Propionibacterium acnes RT6) were not inhibited by castor oil. Xycrobe cells were grown in liquid culture and plated on agar plates. Castor oil was then added to dotted areas on the plates to examine for inhibition. No growth was inhibited.

[0024] Figure 3 shows that the antioxidant activity of the antioxidant concentrate produced by Xycrobe (Propionibacterium acnes roxP) fermentation is stable over 84 days. Detailed Implementation

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any terms and techniques described herein relating to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well-known and commonly used in the art. In the event of any conflict, this disclosure, including the definitions, shall prevail. Exemplary methods and materials are described below, although methods and materials similar to or equivalent to those described herein may be used to practice or test the embodiments and aspects described herein.

[0026] As used herein, the term "Xycrobe" or "Xycrobes" refers to a genetically modified strain of *Propionibacterium acnes* (also known as *Propionibacterium acnes*) that has stopped growing. This strain is described in U.S. Patent No. 10,5843,44B2 and International Patent Application Publication No. WO2015195845A, both of which are incorporated herein by reference to such teachings.

[0027] As used herein, the terms "amino acid," "nucleotide," "polynucleotide," "carrier," "peptide," and "protein" have the common meanings understood by one of ordinary skill in the art. Standard single-letter nucleotides (A, C, G, T, U) and standard single-letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0028] As used herein, terms such as “comprising,” “including,” “containing,” “containing,” “having,” etc., mean “comprising.” This disclosure also considers other embodiments or elements of “comprising,” “consisting of,” and “substantially consisting of,” whether or not explicitly stated.

[0029] The terms “a,” “the,” “the,” and “the,” as used herein, and similar terms used in the context of the disclosure (particularly in the context of the claims), shall be construed as covering both the singular and the plural, unless otherwise stated herein or clearly contrary to the context. Furthermore, “a,” “the,” or “the” means “one or more,” unless otherwise stated.

[0030] As used in this article, the term "or" can be a conjunction or a non-conjunction.

[0031] As used in this article, the term “basically” means to a great extent or in a significant way, but not entirely.

[0032] As used herein, the term "about" or "approximately" applies to one or more values ​​of interest, referring to a value similar to the reference value, or within an acceptable margin of error for a particular value as determined by one of ordinary skill in the art, depending in part on how the value is measured or determined, such as limitations of the measurement system. In one aspect, the term "about" means any numerical value, including integer and fractional components, that varies by no more than ±10% of the numerical value modified by the term "about". Additionally, according to practice in the art, "about" may refer to a value within 3 or more standard deviations. Furthermore, for example, with respect to biological systems or processes, the term "about" may refer to a value within an order of magnitude, within 5 times in some embodiments, and within 2 times in others. As used herein, the symbol "~" means "about" or "approximately".

[0033] All ranges disclosed herein include the endpoints as discrete values ​​and all integers and fractions specified within the range. For example, the range 0.1–2.0 includes 0.1, 0.2, 0.3, 0.4…2.0. If the endpoints are modified by the term “about”, the specified range is expanded to vary by ±10% or 3 or more standard deviations of any value within the range, including the endpoints.

[0034] As used herein, the term "active ingredient" or "active pharmaceutical ingredient" refers to a pharmaceutical preparation, active ingredient, compound or substance, composition or mixture thereof that provides a pharmacological effect (usually beneficial).

[0035] Here, the terms "control" or "reference" are used interchangeably. A "reference" or "control" level can be a predetermined value or range that is used as a baseline or benchmark for evaluating measurement results. "Control" also refers to a control experiment or control cells.

[0036] As used herein, the term “dosage” means any form of active ingredient formulation or composition, including cells, in an amount sufficient to initiate or produce a therapeutic effect by at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.

[0037] As used herein, the term “prevention” means preventing or reducing the development of a disease, whether to a statistically significant degree or to a degree that is detectable by a person skilled in the art.

[0038] As used herein, the term "effective dose" or "therapeutic effective dose" means administering to a subject a substantially nontoxic but adequate amount of a preparation, composition, or cell to prevent, treat, or to some extent improve one or more symptoms of a disease or condition that the subject is experiencing or susceptible to. The result may be a reduction or alleviation of the signs, symptoms, or cause of the disease, or any other anticipated alteration of the biological system. Effective doses may be based on individual factors for each subject, including but not limited to the subject's age, body size, type or severity of disease, stage of disease, route of administration, type or extent of complementary therapy used, ongoing disease process, and type of treatment required.

[0039] As used herein, the term "subject" refers to an animal. Typically, a subject is a mammal. A subject also refers to primates (e.g., humans, males or females; infants, adolescents, or adults), non-human primates, rats, mice, rabbits, pigs, cattle, sheep, goats, horses, dogs, cats, fish, birds, etc. In one implementation, the subject is a primate. In another implementation, the subject is a human.

[0040] As used in this article, a subject is considered "in need of treatment" if they would benefit biologically, medically, or in terms of quality of life from such treatment. Those in need of treatment may not necessarily experience symptoms, especially in the case of preventative or preventative care.

[0041] As used herein, the terms “inhibition,” “inhibitory,” or “inhibitory” refer to the reduction or inhibition of a biological process, condition, symptom, disorder, or disease, or a significant reduction in the baseline activity of a biological activity or process.

[0042] As used herein, “treatment” or “therapeutic” means prevention, aversion, inhibition, suppression, reversal, mitigation, improvement, or inhibition of biological processes, including symptoms or disease progression, or complete elimination of disease. Treatment can be carried out in an acute or chronic manner. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with it before suffering from it. “Inhibition” or “improvement” of a disease, symptom, or its symptoms involves administering the cells, compositions, or compounds described herein to a subject after the clinical onset of such disease, symptom, or its symptoms. “Prevention” or “avoidance” of a disease, symptom, or its symptoms involves administering the cells, compositions, or compounds described herein to a subject before the onset of such disease, symptom, or its symptoms. “Inhibition” of a disease or symptom involves administering the cells, compositions, or compounds described herein to a subject after the onset of the disease or symptom but before its clinical manifestation or symptom presentation.

[0043] Unless otherwise stated, all percentages (%) used in this document refer to mass (or weight, w / w) percentages.

[0044] This article describes a dermatological composition containing probiotic components and dormant genetically modified organisms (GMOs) that can mitigate the harmful effects of pathogenic bacteria and enhance the skin's natural microbiome. The GMO *Propionibacterium acnes* is symbiotic on human skin and can provide cosmetic and therapeutic benefits by: regulating sebum secretion to even out skin tone; secreting antioxidants; reducing oxidative stress that accelerates aging; and soothing the skin to promote a more even texture and reduce pore size. The GMOs are engineered to penetrate the epidermis and enter the dermis. In the dermis, they consume sebum produced by the sebaceous glands while also releasing antioxidants and anti-inflammatory mediators to prevent the growth of pathogenic bacteria and reduce their harmful effects. This process ultimately allows beneficial bacteria to flourish, aiding in skin healing. The composition described herein combats acne by symbiotically enhancing the skin's natural microbiome.

[0045] One embodiment described herein is a dermatological composition comprising one or more growth-arrested genetically modified bacteria or probiotic ingredients; and one or more dermatologically acceptable excipients. In one embodiment, the formulation contains growth-arrested genetically modified bacteria (e.g., Propionibacterium acnes) in an anhydrous or substantially anhydrous composition. The absence of water in the composition is necessary to maintain the bacteria in a dormant state. These bacteria remain dormant until the product is applied to the skin, at which point they encounter moisture and become active. These bacteria are stable in the formulation for at least 6 months, potentially up to 2 years.

[0046] The compositions described herein containing growth-arrested transgenic bacteria unexpectedly contain some ingredients previously considered bactericidal or harmful to bacteria. For example, some compositions contain castor oil, vitamin A, or vitamin A analogs. These compounds are considered harmful to bacteria. See, for example, Al-Mamun et al., BMC Comp. Alt. Med. 16: 211 (2016); Momoh et al., Bul. Envir. Pharm. Life Sci. 1: 21-27 (2012); Rampadarath and Puchoo, Asian Pac. J. Trop. Biomedicine 6(2): 100-107 (2016). Therefore, it is surprising and unexpected that *Propionibacterium acnes* still survives in the formulation in the presence of these potentially bactericidal compounds. See Figure 2. Without being bound by any theory, it is assumed that the bacteria are resistant to any effects of the potentially bactericidal compounds in the formulation because they are in a dormant state.

[0047] In one respect, the composition comprises the formulation shown in Table 1.

[0048]

[0049] On the other hand, the composition comprises the formulation shown in Table 2.

[0050]

[0051] On the other hand, the composition comprises the formulation shown in Table 3.

[0052]

[0053] In another aspect, the composition comprises the formulation shown in Table 4.

[0054]

[0055] Dermatologically acceptable excipients useful to the compositions described herein may further comprise any one or a combination of the following: acidifiers (acetic acid, glacial acetic acid, citric acid, fumaric acid, hydrochloric acid, diluted hydrochloric acid, malic acid, nitric acid, phosphoric acid, diluted phosphoric acid, sulfuric acid, tartaric acid); alkalizing agents (ammonia, ammonium carbonate, diethanolamine, diisopropanolamine, potassium hydroxide, sodium bicarbonate, sodium borate, sodium carbonate, sodium hydroxide, teratamine); and defoamers (dimethylsiloxane). Alkane, siloxane); Antibacterial preservatives (benzalkonium chloride, benzalkonium chloride solution, phenethyl ammonium chloride, benzoic acid, benzyl alcohol, butylbenzoic acid, chloropyridine, chlorobutanol, chlorocresol, cresol, dehydroacetic acid, ethylbenzoic acid, methylbenzoic acid, sodium methylbenzoate, phenol, phenylethanol, phenylmercuric acetate, phenylmercuric nitrate, potassium benzoate, potassium sorbate, acrylic acid, sodium acrylic acid, sodium benzoate, sodium dehydroacetate, sodium propionate, ascorbic acid, thimerosal, thymol). Antioxidants (ascorbic acid, ascorbate palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphite, monothioglycerol, propyl gallate, sodium formaldehyde sulfite, sodium metabisulfite, sodium thiosulfate, sulfur dioxide, tocopherol, tocopherol excipients). Buffers (acetic acid, ammonium carbonate, ammonium phosphate, boric acid, citric acid, lactic acid, phosphoric acid, potassium citrate, potassium metaphosphate, potassium dihydrogen phosphate, sodium acetate, sodium citrate, sodium lactate solution, dibasic sodium phosphate, monobasic sodium phosphate, phosphate buffered saline). Chelating agents (disodium edetate, ethylenediaminetetraacetic acid and its salts, edetate). Coating agents (sodium carboxymethyl cellulose, cellulose acetate, cellulose acetate phthalate, ethyl cellulose, gelatin, pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, methacrylic acid copolymer, methylcellulose, polyvinyl acetate phthalate, shellac, sucrose, titanium dioxide, palm wax, microcrystalline wax, zein). Coloring agents (caramel, red, yellow, black or mixed colors, iron oxide); complexing agents (ethylenediaminetetraacetic acid and its salts (EDTA), acetic acid, glutaric acid ethanolamide, oxyquinoline sulfate); desiccants (calcium chloride, calcium sulfate, silicon dioxide). Emulsifiers and / or solubilizers (gum arabic, cholesterol, diethanolamine (auxiliary agent), glyceryl monostearate, lanolin alcohol, mono- and diglycerides, monoethanolamine (auxiliary agent), lecithin, oleic acid (auxiliary agent), oleyl alcohol (stabilizer), polyoxyethylene 50 stearate, polyoxyethylene 35 castor oil, polyoxyethylene 40 hydrogenated castor oil, polyoxyethylene 10 oleyl alcohol ether, polyoxyethylene 20 hexadecyl ether, polyoxyethylene 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, diacetate, monostearate, sodium lauryl sulfate, sodium stearate, sorbitol monolaurate, sorbitol monooleate, sorbitol monostearate, stearic acid, terbutaline, emulsifying wax).Filter aids (cellulose powder, pure silica); fragrances and perfumes (anisole, benzaldehyde, ethyl vanillin, menthol, methyl salicylate, monosodium glutamate, neroli oil, peppermint, peppermint oil, peppermint extract, rose oil, rose water, thyme, turmeric tincture, vanilla, vanilla tincture, vanillin). Wetting agents (glycerol, hexanediol, sorbitol); plasticizers (e.g., castor oil, diacetylated monoglycerides, diethyl phthalate, glycerol, mono- and diacetylated monoglycerides, propylene glycol, triacetyl, triethyl citrate); polymers (e.g., cellulose acetate, alkyl cellulose, hydroxyalkyl, acrylic polymers and copolymers); solvents (acetone, alcohol, diluted alcohol, propylene hydrate, benzyl benzoate, butanol, carbon tetrachloride, chloroform, corn oil, cottonseed oil, ethyl acetate, glycerol, hexanediol, isopropanol, methanol, dichloromethane, methyl isobutyl ketone, mineral oil, peanut oil, propylene carbonate, sesame oil, water for injection, sterile water for injection, sterile water for irrigation, purified water); adsorbents (cellulose powder, charcoal, pure silica); carbon dioxide adsorbents (barium hydroxide lime, soda lime). Hardeners (hydrogenated castor oil, cetyl alcohol, hexadecyl alcohol, hexadecyl ester wax, stearin, paraffin, polyethylene excipients, stearyl alcohol, emulsion wax, white wax, yellow wax). Suspension agents and / or thickeners (acacia, agar, alginic acid, aluminum monostearate, bentonite, purified bentonite, magmatic bentonite, carbomer, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose, sodium carboxymethyl cellulose, carrageenan, microcrystalline and sodium carboxymethyl cellulose; dextrin, gelatin, guar gum, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, magnesium aluminum silicate, methyl cellulose, pectin, polyethylene oxide, polyvinyl alcohol, povidone, alginate, silica, colloidal silica, sodium alginate, xanthan gum); sweeteners (aspartame, dextrin, glucose, excipient glucose, fructose, mannitol, saccharin, calcium saccharin, sodium saccharin, sorbitol, sorbitol solution, sucrose, compressible sugar, confectionery sugar, syrup); surfactants (silicone). Binders (gum arabic, alginate, sodium carboxymethyl cellulose, microcrystalline cellulose, dextrin, ethyl cellulose, gelatin, liquid glucose, guar gum, hydroxypropyl methylcellulose, methylcellulose, polyethylene oxide, povidone, pregelatinized starch, syrup). Diluents (calcium carbonate, dibasic calcium phosphate, tribasic calcium phosphate, calcium sulfate, microcrystalline cellulose, cellulose powder, dextrin, glucose excipients, fructose, kaolin, lactose, mannitol, sorbitol, starch, pregelatinized starch, sucrose, compressible sugar, confectionery sugar). Disintegrants (alginic acid, microcrystalline cellulose, croscarmellose sodium, croscarmellose, polacolin potassium, sodium starch alkyd, starch, pregelatinized starch).Lubricants (calcium stearate, glyceryl behenate, magnesium stearate, light mineral oil, sodium stearoyl fumarate, stearic acid, refined stearic acid, talc, hydrogenated vegetable oil, zinc stearate); thickeners (gelatin with a swelling strength of 50-100); tonics (glucose, glycerin, mannitol, potassium chloride, sodium chloride); carriers: flavoring agents and / or sweeteners (aromatic solutions, compound benzaldehyde solutions, isool solutions, peppermint water, sorbitol solutions, syrups, toluene syrup); carriers: oily (almond oil, corn oil, cottonseed oil, ethyl oleate, isopropyl myristate, isopropyl palmitate, mineral oil, light mineral oil, myristol, octyl dodecanol, olive oil, peanut oil, persimmon oil, sesame oil, soybean oil, squalane); carriers: solid carriers (sugar spheres); Ingredients: Sterile (antibacterial water for injection, antibacterial sodium chloride injection); thickener (see suspension); water repellent (cyclomethylsiloxane, dimethylsiloxane, silicone oil); and / or solubilizer (benzalkonium chloride, phenethylammonium chloride, acetylpyridine chloride, sodium polychloride, nonylphenyl alcohol ether 9, nonylphenyl alcohol ether 10, octylphenyl polyol 9, polypoloxam, polyoxyethylene 35 castor oil, polyoxyethylene 40, hydrogenated castor oil, polyoxyethylene 50 stearate, polyoxyethylene 10 oleyl alcohol ether, polyoxyethylene 20, hexadecyl ether, polyoxyethylene 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium lauryl sulfate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, tyloxacillin). This list is not intended to be exclusive, but rather represents the categories of excipients and the specific excipients that can be used in the formulations described herein. See Remington's Essentials of Pharmaceutics, Pharmaceutical Press Publishing Company, London, UK, 1. st Edition, 2013, and Handbook of Pharmaceutical Excipients, 8 th Edition, Pharmaceutical Press Publishing Company, London, UK, 2017. These teachings are incorporated herein by reference.

[0056] Another embodiment described herein is an anhydrous dermatological composition comprising: about 0.1% to about 5% (by weight) of one or more wild-type or growth-arrested transgenic bacteria or probiotic components; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable anhydrous excipients. In one aspect, the dermatologically acceptable anhydrous excipients include one or more emollients, humectants, moisturizers, vitamins, vitamin analogs, gelling agents, rheology modifiers, pH adjusters, or preservatives; and optionally one or more solvents.

[0057] Another embodiment described herein is an anhydrous dermatological composition comprising: one or more wild-type or growth-arrested transgenic microorganisms; and one or more dermatologically acceptable anhydrous excipients, including one or more emollients, humectants, vitamins, vitamin analogs, or gelling agents. In one aspect, the anhydrous dermatological composition maintains the growth-arrested transgenic microorganisms in a dormant state for at least 6 months and up to about 2 years.

[0058] In one embodiment, the microorganism may include organisms of the genus Cutibacterium. In another embodiment, the microorganism includes those selected from *Cutibacterium acidifaciens*, *Cutibacterium acidipropionici*, *Cutibacterium acnes*, *Cutibacterium australiense*, *Cutibacterium avidum*, *Cutibacterium cyclohexanicum*, *Cutibacterium freudenreichii*, *Cutibacterium granulosum*, *Cutibacterium jensenii*, *Cutibacterium microaerophilum*, *Cutibacterium propionicum*, or *Cutibacterium thoenii*. In one embodiment, the microorganism is *Cutibacterium acnes*. In one embodiment, *Propionibacterium acnes* comprises a CRISPR (clustered, regularly spaced short palindromic repeats) array. In one embodiment, the microorganism is *Propionibacterium acnes* type II, type 6 ribosomes (e.g., *Propionibacterium acnes* RT6). In another embodiment, the microorganism is *Propionibacterium acnes* roxP.

[0059] Another embodiment described herein is an anhydrous dermatological composition comprising: about 0.1% to about 5% (by weight) of growth-arrested acne-causing keratinocytes; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable anhydrous excipients, including one or more emollients, humectants, vitamins, vitamin analogs, or gelling agents. In one aspect, the anhydrous dermatological composition maintains growth-arrested acne-causing keratinocytes in a dormant state for at least 6 months and up to about 2 years. In another aspect, the composition comprises: about 1% to 2% (by weight) of growth-arrested acne-causing keratinocytes; about 90% (by weight) of one or more emollients; and about 10% (by weight) of one or more gelling agents. In yet another aspect, the growth-arrested acne-causing keratinocytes comprises: a lyophilized powder comprising acne, sodium chloride, calcium stearate, and inulin (fructosaccharide polysaccharide). In another aspect, the emollient includes ethyl olive oil, olive leaf extract, and castor oil; the gelling agent includes polyamide-3 (dilinoleic acid, ethylenediamine, PEG / PPG-32 / 10 aminopropyl methyl ether-terminated polypropylene glycol diamine). In another aspect, the composition includes: about 0.1-5% (by weight) of growth-arrested acne-causing keratinocytes; about 55-95% (by weight) of ethyl olive oil and olive leaf extract; about 20-40% (by weight) of castor oil; and about 1-15% (by weight) of polyamide-3 (dilinoleic acid, ethylenediamine, PEG / PPG-32 / 10 aminopropyl methyl ether-terminated polypropylene glycol diamine). In another aspect, the composition includes: about 1% to 2% (by weight) of growth-arrested acne-causing keratinocytes; about 1% (by weight) of vitamin A or a vitamin A analogue; about 90% (by weight) of one or more emollients; and about 10% (by weight) of one or more gelling agents. On the other hand, the composition comprises: about 0.1-5% (by weight) of growth-arrested acne keratinocytes; about 0.1-5% (by weight) of retinol; about 0.1-5% (by weight) of vitamin A palmitate; about 50-75% (by weight) of ethyl olive oil and olive leaf extract; about 20-40% (by weight) of castor oil; and about 1-15% (by weight) of polyamide-3 (dilinoleic acid, ethylenediamine, PEG / PPG-32 / 10 aminopropyl methyl ether-terminated polypropylene glycol diamine).

[0060] Another embodiment described herein is an aqueous dermatological composition comprising: about 0.1% to about 5% (by weight) of a probiotic composition; and about 95% to about 99.9% (by weight) of one or more dermatologically acceptable excipients, including one or more humectants, moisturizers, rheology modifiers, pH adjusters, preservatives, or solvents. In one aspect, the probiotic composition includes a *Propionibacterium acnes* fermentation product comprising: a lyophilized powder of purified *Propionibacterium acnes* roxP fermentation medium. In another aspect, the acne fermentation product is purified by filtration and double filtration. In yet another aspect, the composition comprises: about 0.1% to 5% (by weight) of the *Propionibacterium acnes* fermentation product; about 80% (by weight) of one or more solvents; about 10% (by weight) of one or more humectants; about 1% (by weight) of one or more moisturizers; about 2% (by weight) of one or more rheology modifiers; about 4% (by weight) of one or more film-forming agents; about 2% (by weight) of one or more preservatives; and about 1% (by weight) of one or more pH adjusters. On the other hand, the composition comprises: about 0.1-5% (by weight) of *Propionibacterium acnes* fermentation product; about 70-90% (by weight) of water; about 0.1-5% (by weight) of sodium hyaluronate solution; about 5-15% (by weight) of acetamide ethoxyethanol; about 1-5% (by weight) of polyurethane; and about 0.1-5% (by weight) of a solution of sodium protocatechuate, sodium lactate, protocatechuic acid, arginine, aspartic acid, glycine, alanine, serine, valine, proline, threonine, isoleucine, histidine, and phenylalanine. The composition comprises about 0.1-5% (by weight) of a copolymer of hydroxyethyl acrylate and sodium acryloyldimethyl taurate; about 0.1-5% (by weight) of polyacrylate crosspolymer-6; about 0.1-5% (by weight) of benzyl alcohol; about 0.5-1.5% (by weight) of sodium benzoate and potassium sorbate; and about 0.1-5% (by weight) of a citric acid solution (50%). In another aspect, the composition further comprises about 0.1% to about 2% (by weight) of retinoic acid.

[0061] Another embodiment described herein is a method for manufacturing the composition described herein. In one aspect, the method includes combining the components of the composition. These components may be combined in any order or at specific stages. For example, components of one stage may be combined, and components of any other stage may be combined, and then the two separate stages may be combined and mixed to a homogeneous state. Heating and mixing may be used alone or in combination to promote homogenization.

[0062] Another embodiment described herein is a cosmetic or therapeutic composition prepared by any of the methods or means described herein.

[0063] Another embodiment described herein is a method for preparing a probiotic fermentation product, the method comprising: culturing microorganisms in a fermentation medium; separating the fermentation medium from the organisms; performing tangential flow filtration on the fermentation medium exchanged with a buffer to remove small molecule contaminants, producing a purified fermentation product; and freeze-drying the purified fermentation product. In one aspect, the microorganism is *Propionibacterium acnes* (C. acnes rox P).

[0064] Another embodiment described herein is a fermentation product produced by any of the methods or means described herein.

[0065] It will be apparent to those skilled in the art that appropriate modifications and adjustments can be made to the compositions, formulations, methods, processes, and applications described herein without departing from the scope of any embodiment or aspect thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any particular embodiment. All the various embodiments, aspects, and options disclosed herein can be combined in any variation or iteration. The scope of the compositions, formulations, methods, and processes described herein includes all actual or potential combinations of the embodiments, aspects, options, examples, and preferences described herein. Any composition, formulation, or method described herein may omit any ingredient or step, substitute for any ingredient or step disclosed herein, or include any ingredient or step disclosed elsewhere herein. The mass ratio of any component of any composition or formulation disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation is disclosed herein as if explicitly disclosed. If the meaning of any term in any patent or reference conflicts with the meaning of the term used in this disclosure, the meaning of the term or phrase in this disclosure shall prevail. Furthermore, the embodiments disclosed and described herein are merely exemplary. All patents and publications referenced herein are incorporated by reference to their specific doctrines.

[0066] Example

[0067] Example 1

[0068] The formulation of the live Xycrobe probiotic gel consists of four (4) ingredients, including a genetically modified live strain of Propionibacterium acnes whose growth has been halted. The formulation is shown in Table 5.

[0069]

[0070] The preparation method of this formula is as follows.

[0071] 1. Add phase B together and heat to 90-100℃ while stirring;

[0072] 2. During heating in the "B" phase, Xycrobe powder is dispersed into ethyl oleate and olive leaf extract by high shear force mixing. In Light ET), the "A" phase is formed;

[0073] 3. After all the solids have dissolved in the "B" phase, the solution is cooled to solid state;

[0074] 4. Mix phase "A" and phase "B" together.

[0075] The live probiotic gel formulation disclosed in Table 5 is the carrier of choice for delivering the Xycrobe strain of *Propionibacterium acnes* (formerly *Propionibacterium acnes*) to the skin. This carrier is an anhydrous gel with shear-thinning properties. An anhydrous formulation is required to extend the product's shelf life and prevent Xycrobe from multiplying in the matrix due to the lack of water. A polyamide-3 structurer (20,000 Da) traps the emollient oils within the formulation through a hydrophobic pocket, while the polymers are locked together by hydrogen bonds. Therefore, the product maintains a viscous gel structure, suspending the freeze-dried Xycrobe. Once sufficient shear force is applied to the carrier, the structurer breaks down, releasing the emollient oils and allowing the formulation to spread on the skin. Another benefit of the polyamide-3 structurer is the formation of a waterproof film on the skin, which helps retain skin moisture and allows the freeze-dried Xycrobe to spread. LightET is used as the primary emollient in the base because it provides a light and dry feel on the skin. Castor oil is chosen as a secondary emollient for two reasons. First, it does not dry out the skin; second, as it is a triester of ricinoleic acid and glycerin, it contains multiple hydroxyl (-OH) functional groups, which helps in gel formation.

[0076] The viability of Xycrobe strains in the finished product was screened by serially diluting the gel onto a modified RCM-potato peptone and arabinose medium. This screening process examined the total colony-forming units (CFU) of Xycrobe bacteria in the finished product corresponding to a single daily dose. RT-PCR was performed on the Xycrobes prior to lyophilization to ensure the purity of the Xycrobe raw material strain. The lyophilization process of the Xycrobe powder creates a stable raw material, making it easier to incorporate into anhydrous gels. The Xycrobe powder is a mixture of Xycrobe cells, sodium chloride, calcium stearate, and inulin (fructosaccharide polysaccharide).

[0077] Example 2

[0078] A secondary formulation for live Xycrobe probiotic gel has been developed, incorporating retinoic acid. The preparation of the retinoic acid-containing live Xycrobe probiotic gel is similar to the aforementioned version. This formulation is shown in Table 6.

[0079]

[0080]

[0081] The preparation of live Xycrobe probiotic gel containing retinoic acid is similar to that of the live Xycrobe probiotic gel described above.

[0082] Polyamide-3 (Oleo Craft) TM One drawback of MP-30 (e.g., dilinoleic acid, ethylenediamine, PEG / PPG-32 / 10 aminopropyl methyl ether-terminated polypropylene glycol diamine) structural agents is the difficulty in gelling silicone systems. See Oleo Craft. TM Polymer product information sheet, Croda Europe Ltd (January 2017). While silicones offer the best sensory experience, achieving a stable, transparent, and globally recognized formulation is a challenge. The solution lies in the addition of emollients, ethyl olive oil, and olive leaf extract (…). Light ET). Light ET is a naturally sourced, globally compliant silicone alternative. In addition to providing desirable sensory properties, LightET can also enhance skin hydration and antioxidant activity. See Light ET Product Manual, Hallstar Beauty, March 13, 2019.

[0083] The second major ingredient in Live Xycrobe Probiotic Gel is castor oil. Castor oil has a long history and is known for its antimicrobial activity against several bacteria. For example, see Al-Mamun et al., “Characterization and evaluation of antibacterial and antiproliferative activities of crude protein extracts isolated from the seed of Ricinus communis in Bangladesh,” BMC Comp. Alt. Med. 16: 211 (2016); Momoh et al., “Evaluation of the antimicrobial and phytochemical properties of oil from castor seeds (Ricinus communis linn),” Bul. Envir. Pharm. Life Sci. 1: 21-27 (2012); Rampadarath and Puchoo. “In vitro antimicrobial and larvicidal properties of wild Ricinus communis L. in Mauritius,” Asian Pac. J. Trop. Biomedicine 6(2): 100 > 107 (2016). Pure Seatons virgin castor oil (Croda Personal Care) was found to have no inhibitory effect on the growth of Xycrobe cells, despite numerous studies describing the antimicrobial activity of castor oil. (See Figure 2.) This result was both surprising and unexpected.

[0084] The initial Xycrobe raw material consisted only of inulin and Xycrobe. This mixture was considered impractical for long-term storage or scaling up. The hygroscopic nature of Xycrobe powder made the raw material susceptible to subtle changes in humidity and required use shortly after exposure to the atmosphere. To eliminate the powder's hygroscopicity, 1% calcium stearate and sodium chloride were added to the mixture prior to freeze-drying. Calcium stearate was added as a processing aid (anti-caking agent) to prevent powder clumping and provide a smooth, dried powder. Another benefit of using calcium stearate is that it helps eliminate the roughness of the Xycrobe raw material.

[0085] Example 3

[0086] The third formulation is an antioxidant concentrate derived from the Xycrobe biosphere, containing a purified fermentation medium from Xycrobe (C. acnesrox P) cultures. During Xycrobe fermentation, a complex mixture of nutrients and cellular metabolites is produced. This fermentation product is separated from the Xycrobe cells by filtration (0.22 microns) to obtain a sterile fermentation product.

[0087] The fermentation medium has a strong odor due to the presence of some metabolites. To remove the odor, the medium is filtered sequentially and concentrated using percolation. In a tangential flow filtration (TFF) system, a truncated cellulose or similar material filter with a strength of 3 kDa or less is used to concentrate the fermentation medium. All filtration steps are performed at 4°C or 25°C. After samples have been prepared that have been concentrated at least 20 times, a modified percolation process is performed for this purpose. By exchanging the concentrate with PBS buffer, residual low-molecular-weight components causing the odor are removed, yielding only substances with antioxidant and anti-inflammatory properties.

[0088] The fermentation products (antioxidant concentrate derived from the Xycrobe biota) were stored as a concentrate.

[0089] The main ingredient in the Xycrobe bio-derived antioxidant concentrate is rose water. Rose water was chosen to help mask the odor of the Xycrobe fermentation products and for their associated beneficial properties. However, it was possible to devise ways to remove the odor of the fermentation products, as described herein. Therefore, the amount of rose water could be reduced or eliminated. Since this is a water-based system, a preservative was required to prevent microbial contamination. Several ingredients were screened to identify a preservative that would effectively prevent microbial contamination without impairing the application of live Xycrobe to the skin using other Xycrobe products recommended by the Xycrobe protocol. The preservative chosen was 0.1-5% benzyl alcohol. Benzyl alcohol is well tolerated on the skin and effective over a wide pH range. Two polymers were used (polyacrylate crosspolymer-6, e.g., Sepimax). TM Zen) and hydroxyethyl acrylate / sodium acryloyl dimethyl taurate copolymer (e.g., Sepinov) TM Combine EMT10 in a 1:1 ratio and thicken the system into a gel. The formulation is shown in Table 7.

[0090]

[0091] In another embodiment, the compositions shown in Table 7 further comprise fat-soluble vitamins, including vitamin A (retinol), vitamin A analogs (e.g., vitamin A palmitate, paraquat), vitamin E, vitamin D, or vitamin K, or analogs thereof. In one aspect, the compositions shown in Table 7 further comprise retinol or retinone in a weight percentage of about 0.1% to about 2%, including all integers within that range.

[0092] Table 7 shows the formulation of the antioxidant concentrate derived from the Xycrobe biota. The preparation method of this formulation is as follows.

[0093] 1. Mix the components of phase "A" together.

[0094] 2. Add "B" phase until the pH of the system is 4.5-5.0 to prepare "AB" phase.

[0095] 3. The "C" phase component is slowly added to the "AB" phase to form the "ABC" phase; and

[0096] 4. The "D" phase is added to the "ABC" mixture in sufficient quantity.

[0097] Using OxiSelec TM The Trolox Equivalent Antioxidant Capacity (TEAC) assay kit quantifies the antioxidant activity of an antioxidant concentrate derived from the Xycrobe biota. The TEAC assay measures the amount of antioxidant according to Trolox standards. Trolox is an antioxidant (a vitamin E derivative). The standardized method for the TEAC kit involves adding oxidized ABTS substrate to Trolox and measuring the absorbance of the solution. As the substrate is reduced, the solution becomes clear and the absorbance decreases. Samples were obtained after storage at room temperature for 14, 26, and 84 days, and antioxidant activity was measured over time. Figure 3 shows that the antioxidant activity of the concentrate was stable over 84 days.

Claims

1. An anhydrous therapeutic dermatological composition comprising: 0.1% to 5% of the mass of stagnant acne-causing keratinobacteria ( Cutibacteriumacnes A transgenic strain that can remain dormant and stable for at least 6 months to 2 years; One or more anhydrous emollients, comprising ethyl olive oil or olive leaf extract, at a weight of 55% to 95%; and One or more anhydrous gelling agents, including polyamide-3, at 1% to 15% by weight.

2. The composition according to claim 1 further comprises 20% to 40% by weight of castor oil.

3. The composition according to claim 2, further comprising: One or more vitamin A, vitamin A analogs, retinoids, or combinations thereof, at a weight of 0.1% to 5%.

4. The composition of claim 1, wherein the composition comprises: 1% to 2% of the quality of genetically modified strains of *Propionibacterium acnes* with growth retardation; One or more anhydrous emollients with 90% mass; as well as One or more anhydrous gelling agents at 10% by weight.

5. The composition of claim 1, wherein the growth-arrested *Propionibacterium acnes* transgenic strain comprises a lyophilized powder containing *Propionibacterium acnes*, sodium chloride, calcium stearate, and inulin.

6. Use of an anhydrous therapeutic skin composition in the preparation of a pharmaceutical agent for enhancing the skin microbiome, said anhydrous skin composition comprising: 0.1% to 5% by weight of a growth-arrested genetically modified strain of *Keratinobacterium acnes*, which remains dormant and stable for at least 6 months to 2 years; 55% to 95% by weight of one or more anhydrous emollients, including ethyl olive oil or olive leaf extract; and 1% to 15% by weight of one or more anhydrous gelling agents, including polyamide-3.

7. Use of an anhydrous therapeutic skin composition in the preparation of a pharmaceutical agent for minimizing the growth of pathogenic bacteria on the skin, said anhydrous therapeutic skin composition comprising: 0.1% to 5% by weight of a growth-arrested genetically modified strain of *Keratinobacterium acnes*, which remains dormant and stable for at least 6 months to 2 years; 55% to 95% by weight of one or more anhydrous emollients, including ethyl olive oil or olive leaf extract; and 1% to 15% by weight of one or more anhydrous gelling agents, including polyamide-3.

8. Use of an anhydrous therapeutic skin composition in the preparation of a medicament for treating acne, said anhydrous therapeutic skin composition comprising: 0.1% to 5% by weight of a growth-arrested genetically modified strain of *Keratinobacterium acnes*, which remains dormant and stable for at least 6 months to 2 years; 55% to 95% by weight of one or more anhydrous emollients, including ethyl olive oil or olive leaf extract; and 1% to 15% by weight of one or more anhydrous gelling agents, including polyamide-3.

9. A method for preparing an anhydrous therapeutic skin composition comprising: 0.1% to 5% by weight of a growth-arrested transgenic strain of *Propionibacterium acnes*, which remains dormant and stable for at least 6 months to 2 years; 55% to 95% by weight of one or more anhydrous emollients, including ethyl olive oil or olive leaf extract; and 1% to 15% by weight of one or more anhydrous gelling agents, including polyamide-3.

10. Anhydrous therapeutic skin composition prepared by the method according to claim 9.

11. Use of an anhydrous therapeutic skin composition in the preparation of an agent for treating skin diseases or conditions, said anhydrous therapeutic skin composition comprising 0.1% to 5% by weight of a growth-arrested transgenic strain of *Propionibacterium acnes* that remains dormant and stable for at least 6 months to 2 years; 55% to 95% by weight of one or more anhydrous emollients, including ethyl olive oil or olive leaf extract; and 1% to 15% by weight of one or more anhydrous gelling agents, including polyamide-3.

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