Fermentation extracts for providing skin care benefits and methods thereof

Abstract

The present disclosure relates to skin care compositions, skin care products, and methods for providing at least one skin care benefit in a subject in need thereof. More specifically, the present disclosure relates to methods and compositions comprising a Bacillus subtilis ferment, a Bacillus subtilis ferment extract, or a fraction thereof for reducing dandruff, reducing scalp or skin disorders, enhancing skin barrier, improving skin barrier function, moisturizing skin, and any combination thereof.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 509620, filed June 22, 2023, and U.S. Provisional Application No. 63 / 567144, filed March 19, 2024, each of which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to skin care compositions, skin care products, and methods for providing at least one skin care benefit in subjects in need. More specifically, this disclosure relates to methods and compositions comprising Bacillus subtilis ferment, Bacillus subtilis ferment extract, or fractions thereof for reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof. References to sequence lists submitted electronically

[0003] The contents of the sequence list, which was submitted electronically with the application in an XML file (name: 20240617_NB42207PCT_SequenceListing.xml; size: 72,375 bytes; created on: June 17, 2024), form part of this application and are hereby incorporated herein by reference in their entirety. Background Technology

[0004] The skin acts as a barrier, preventing organisms from drying out and protecting them from the penetration of external (often harmful) substances. The skin barrier is vital to human life because it physically protects against external threats such as infectious agents, chemicals, systemic toxicities, and allergens, and internally helps maintain skin homeostasis and protects against increased water loss from the body (Skin barrier function, 2016, T. Agner, ed., Karger, Basel, Switzerland, 164 pages, ISBN 978-3-318-05585-6).

[0005] Human skin consists of two main cellular layers: the epidermis and the dermis. The epidermis forms the outermost layer of the skin and is mainly composed of terminally differentiated keratinocytes and lipids, with living, dividing keratinocytes located beneath the terminally differentiated keratinocytes. The outermost layer of the epidermis (stratum corneum or horny layer) is the part in contact with the environment, and the specific structure of the horny layer protects the skin and stabilizes its flexibility by binding a certain amount of water (PM Elias, Structure and Function of the Stratum Corneum Permeability Barrier, Drug Dev. Res. 13, 1988, 97-105). The skin barrier function largely depends on the structure and composition of the stratum corneum, which consists of flattened, anucleate cells surrounded by a highly organized and continuous lipid matrix (Skin barrier function, 2016, T. Agner, ed., Karger, Basel, Switzerland, 164 pages, ISBN 978-3-318-05585-6). The epidermis's primary function is to form a permeable barrier against environmental challenges (such as UV radiation, heat, chemicals, and pollution) and pathogens (such as bacteria, fungi, parasites, and viruses). It also prevents the body from uncontrolled evaporation of water from the inside out, thus maintaining hydration balance and skin metabolism.

[0006] The stratified epithelium of the skin is composed of multiple layers of keratinocytes in various stages of differentiation. These layers, from the outermost to the bottom, are generally referred to as the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. In general, these layers endow the skin with a variety of functions, among which providing a physical barrier against the external environment and microorganisms is the primary function (Hogan et al., 2012, Journal of Allergy 2012:901940:1-7). It is widely assumed that a defective skin barrier can lead to a number of skin disorders, including psoriasis, atopic dermatitis, seborrheic dermatitis and dandruff (Hogan et al., 2012, Journal of Allergy 2012:901940:1-7; Sano, Shigetoshi, 2015, Dermatologica Sinica 33(2):64-69; Wikramanayake TC et al., 2019, Experimental Dermatology 28(9):991-1001), as well as dry skin and skin irritation.

[0007] As keratinocytes differentiate, they express a large number of proteins, enzymes, and lipids involved in the formation of the stratum corneum, which is the skin's first line of defense. Essential characteristics of the stratum corneum that contribute to the integrity of the skin barrier include cross-linked structural proteins, lipids, and tightly connected intercellular adhesions. Genetic mutations that impair the production of these components are associated with many skin disorders, demonstrating that all barrier components are important for maintaining skin health (Eckert, RL and E. A. Rorke, 1989, Environmental Health Perspectives 80:109-116; O'Regan G. M. et al., 2009, 124(3):R2-R6; Capon F., 2017, International Journal of Molecular Sciences 18(12):2526; Jennemann E. et al., 2012, Human Molecular Genetics 21(3):586-608; Deak F. et al., 2019, Front. Cell. Neurosci. 2019:13; Lee JYW and JAM McGrath, 2021, British Journal of Dermatology). [British Journal of Dermatology] 184(4): 596-605.

[0008] Closely packed keratinocytes in the stratum corneum are a feature that maintains the integrity of the skin barrier, thereby limiting water loss and preventing the translocation of microbial pathogens and environmental pollutants (Bazzoni G. and E. Dejana, 2002, J. Cell Biology 156(6):947-949; Johnson J. et al., 2014, ColdSpring Harbor Perspectives in Medicine 4(11)). The expression of a series of transmembrane proteins is a strategy used by keratinocytes to connect together. Three transmembrane systems that are important for ensuring that adjacent keratinocytes are tightly connected are desmosomes, cadherin (Cdh), and protocadherin (Pcdh). Desmosomes (intracellular adhesion junctions that connect the intermediate filaments of adjacent cells) are composed of key proteins such as desmoplanin (Dsg), desmocollin (Dsc), and desmoplankin (DP). Disruption of desmosome formation can lead to loss of skin barrier integrity (Lee JYW and JAMcGrath, 2021, British Journal of Dermatology 184(4): 596-605; Johnson J. et al., 2014, Cold Spring Harbor Perspectives in Medicine 4:(11)). Cadmins and the cadmin subfamily protocadmins, like desmosomes, are key intracellular adhesion junctions connecting actin filaments of adjacent cells. Both represent large classes of proteins, with protocadmins further divided into three subclasses: α, β, and γ. Disruption of cadherin and procadherin function leads to loss of epithelial barrier integrity and can contribute to disease progression (Brandner J. et al., 2010, The OpenDermatology Journal 4:14-20; Pancho A. et al., 2020, Frontiers in Molecular Neuroscience 13:117; Tinkle CL et al., 2008, PNAS 105(40):15405-15410).

[0009] Keratinocytes in the stratum corneum differentiate to form a specialized membrane called the keratin capsule. The keratin capsule is primarily composed of cross-linked structural proteins and lipids and helps maintain the integrity of the skin barrier. Key proteins that give the keratin capsule its function include filin, loricrin, loricrin, late capsule protein (LCE), and sciellin (SCEL). Enzymes are required to cross-link proteins such as loricrin and loricrin, with transglutaminase (TGM) being the most prevalent. Ceramides and long-chain fatty acids are the most abundant lipid components in the stratum corneum and help provide a hydrophobic barrier to maintain water and moisture homeostasis. A class of enzymes called ceramide synthases (CERS) and very long-chain fatty acid elongases (ELOVL) are crucial for the synthesis of these lipids. Ceramides require the addition of sphingolipids to fatty acids, and the production of sphingolipids thus requires enzymes such as FA2H (fatty acid 2-hydroxylase) (Proksch E. et al., 2008, Experimental Dermatology 17: 1063-1072; Uchida Y. et al., 2007, Journal of Biological Chemistry 282(18): 13211-13219).

[0010] A crucial function of the skin barrier is maintaining hydration, which involves a delicate balance of moisture content (Proksch E. et al., 2008, Experimental Dermatology 17: 1063-1072). Normally, the skin barrier prevents excessive water loss and absorption to ensure the skin remains moist and does not dry out. A key component of the hydrating epidermis is hyaluronic acid (HA) (Papakonstantinou E. et al., 2012, DermatoEndicrinolgy 4(3):253-258). For full effectiveness, HA is cross-linked, mediated by enzymes such as TNFAIP6 (Evrard C. et al., 2021, JID Innovations 1:100054). Disruption of the physical barrier (such as that caused by skin abrasions or inflammatory diseases, including but not limited to eczema, psoriasis, and atopic dermatitis) can disrupt this balance of moisture regulation. Therefore, when the physical skin barrier is disrupted, proper functioning of the cellular processes necessary to restore the skin barrier to its intact form is essential. Prolonged exposure of the epidermis and subdermis, as exhibited in conditions such as eczema, psoriasis, atopic dermatitis, contact dermatitis, burns, and ulcers resulting from diabetic complications, vasculitis, and ischemic injuries, can lead to dryness and chronic inflammation due to continuous exposure to microbial pathogens and environmental pollutants (Proksch E. et al., 2008, Experimental Dermatology 17:1063-1072). A common method for measuring skin barrier integrity and associated skin hydration is to measure transepidermal water loss (TEWL). TEWL measurements indicate the amount of water evaporated from the skin surface. Higher TEWL values ​​indicate greater water loss and are therefore associated with drier skin (Proksch E. et al., 2008, Experimental Dermatology 17:1063-1072). Measuring the skin barrier resistance is another method for assessing skin barrier integrity. Higher resistivity values ​​are associated with better barrier integrity, while lower resistivity values ​​are associated with poorer barrier integrity. TEWL values ​​are known to be consistent with resistivity measurements in laboratory / research environments (Guth K., et al., 2015, Toxicology InVitro [In Vitro Toxicology] 29:113-123). Therefore, lower TEWL values ​​can be reflected by higher resistivity values ​​and thus indicate an intact barrier resulting in minimal water loss.

[0011] Current treatments for most skin disorders directly target inflammation or the microbes involved, and when any component is inhibited, these treatments allow the skin to recover and form a complete barrier. For example, the use of biologics (such as monoclonal antibodies) to inhibit cytokines in skin disorders such as psoriasis and atopic dermatitis allows for the normalization of the expression of fimbriae and other barrier components (Jeon C. et al., 2017, Hum. Vaccin. Immunother. 13(10):2247-2259; Bieber T., 2020, European Journal of Allergy and Clinical Immunology 75(1):54-62). Particularly for dandruff, antifungal components in anti-dandruff shampoos, such as zinc pyrithione (ZPT) and octipirox, directly control the overgrowth of various Malassezia species. Reducing the overgrowth of Malassezia reduces scalp inflammation and subsequently leads to the restoration of the skin barrier (Warner, RR, et al., 2001, Journal of the American Academy of Dermatology 45(6): 897-903). Other treatments such as salicylic acid and coal tar help remove dead skin (Ranganathan, S. and T. Mukhopadhyay, 2010, Indian Journal of Dermatology 55(2):130-134).

[0012] When tetrachlorophthalide (SENKY), extracted from celery seed, was applied to the scalp of individuals with dandruff, a decrease in dandruff scores, as well as reduced exfoliation and improved transepidermal water loss (TEWL), were observed. Mechanistically, SENKY increases the expression of multiple skin barrier components, including but not limited to scleroderma, filin, SPRR2B, and LCE3B. This study suggests that improving the skin barrier may help reduce dandruff (Mondon, P. et al., 2017, International Journal of Cosmetic Science, 39: 617-621).

[0013] The skin is also home to a diverse community of microbes, most of which are symbiotic (non-pathogenic, permanently resident microbes) or transient (temporarily resident microbes) organisms. In pathogenic interactions, only the microbes benefit, while the host ultimately suffers harm. Typically, many skin pathogens can be found surviving on the skin as symbiotic organisms, but microbial dysbiosis (or microbial imbalance), host genetic variation, and immune status can drive the transition from symbiotic to pathogenic organisms (Findley, K. and Grice, EA, The Skin Microbiome: A Focus on Pathogens and Their Association with Skin Disease. PLoS Pathog. 2014, 10).

[0014] The epidermis constitutes the outermost layer of skin tissue and thus forms an actual protective shell against the environment. The outermost layer of the epidermis (stratum corneum or horny layer) is the part that comes into contact with the environment. The specific structure of the horny layer protects the skin and stabilizes its flexibility by binding a certain amount of water (PM Elias, Drug Dev. Res. [Drug Development Research] 13, 1988, 97-105).

[0015] Spatially, the skin microbiome extends into the subepidermal compartments (Nakatsuji, T et al., The Microbiome Extends to Subepidermal Compartments of Normal Skin. Nat. Commun. [Nature Communications] 2013, 4). Areas with high sebaceous gland density (such as the face, chest, and back) promote the growth of lipophilic microorganisms such as Propionibacterium and Malassezia.

[0016] The skin is a unique environment where microorganisms typically exist in the form of biofilms (Brandwein et al., 2016. NPJ Biofilms Microbiomes 2:3). Biofilms can form on the epithelial surface of the skin or within hair follicles. In addition to cells, biofilms consist of extracellular components such as extracellular polysaccharides, proteins, and DNA. This complex structure can act as a physical and chemical barrier against certain compounds. More importantly, the physiology of microorganisms in the biofilm state differs significantly from that in the planktonic state. This is especially true for their ability to resist environmental stresses and various antimicrobial therapies (Koo et al., Nature Reviews Microbiology 15:740-755, 2017).

[0017] It has been shown that yeast species of the genus *Malassezia* isolated from both healthy and unhealthy skin form biofilms in vitro (Angiolella et al. 2020, Med Mycol. 0:1-7). These isolates of *Malassezia globosa* (M. globosa) can be highly adhesive and / or hydrophobic, and are biofilm producers. Biofilm-formed *Malassezia* species have been shown to have significantly reduced susceptibility to antifungal agents (Figueredo et al., 2016, Medical Mycology 8:863-867, 2013; Bumroogthai et al., Medical Mycology 54:544-549). Biofilm adhesion and hydrophobicity are considered virulence factors of Malassezia (Allen et al., 2018, J. of Clinical & Experimental Dermatology Research 6:311, 2015; Angiolella et al., Medical Mycology 56:110-116). Therefore, strategies to remove Malassezia biofilms may be beneficial for treating various skin conditions caused by this group of organisms.

[0018] Malassezia is a major fungus in the skin microbiome, present on the scalp of almost everyone, and is associated with the most common skin disorders, such as, but not limited to, seborrheic dermatitis, dandruff, and tinea versicolor. Dandruff is a common term used for excessive sebum production on the scalp. It is primarily associated with species of the genus Malassezia, such as *Malassezia restricta* (M. restricta) and *Malassezia spheroidae*, and has a very high prevalence in nearly 50% of the population (Schommer, NN; Gallo, RL, Structure and Function of the Human Skin Microbiome. Trends Microbiol. 2013, 21, 660-668). Improvement of the disorder can be achieved through the therapeutic application of antifungal agents, but not with antibacterial agents. The mechanisms of potential pathogenicity are not fully understood. Impaired skin barrier function can accelerate the progression of the disorder (Harding, C. R et al., Dandruff: a condition characterized by decreased levels of intercellular lipids in scalp stratum corneum and impaired barrier function. Arch. Dermatol. Res. 2002, 294, 221-230).

[0019] Malassezia species lack fatty acid synthases, so they must rely on sebum lipids as a carbon source. They also lack δ2,3-enoyl-CoA isomerases for the efficient utilization of unsaturated fatty acids (e.g., oleic acid). Malassezia species feed on sebum (by secreting one or more lipases that break down triglycerides into irritating fatty acids), and free fatty acids (such as oleic acid) are released as byproducts as sebum breaks down. Many people are sensitive to free fatty acids because they can induce excessive proliferation and dandruff, or induce the release of arachidonic acid, which is also involved in inflammation, and these individuals' scalps will respond to the irritation. In response to the irritation, the scalp begins to become inflamed, red, and itchy, and the body sheds skin cells more quickly than usual in an attempt to remove the irritant. This shedding of skin results in visible scales on the scalp, i.e., dandruff.

[0020] Human skin is colonized by a diverse microbiome (Byrd, A., Belkaid, Y. & Segre, J. The human skin microbiome. Nat Rev Microbiol 16, 143-155; 2018). Staphylococcus hominis, Staphylococcus epidermis, and Cutibacteria acnes (formerly known as Propionibacterium) are known commensal species.

[0021] There remains a need to find methods and skin care compositions for reducing scalp dandruff and scalp or skin barriers. Furthermore, there remains a need to find methods and skin care compositions for strengthening the skin barrier, improving skin barrier function, and moisturizing the skin or scalp. Summary of the Invention

[0022] This disclosure relates to skin care compositions, skin care products, and methods for providing at least one skin care benefit in subjects in need. More specifically, this disclosure relates to methods and compositions comprising Bacillus subtilis ferment, Bacillus subtilis ferment extract, or fractions thereof for reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0023] The inventors unexpectedly observed that the fermentation extract (cell-free supernatant) of Bacillus subtilis (such as, but not limited to, Bacillus subtilis N01, Bacillus subtilis PC01, Bacillus subtilis G01, Bacillus subtilis G01_spo, and Bacillus subtilis K01) reduced the growth of Malassezia species. The inventors also unexpectedly observed that the fermentation extract (cell-free supernatant) of Bacillus subtilis (such as, but not limited to, Bacillus subtilis G01 and Bacillus subtilis G01_spo) not only reduced the growth of Malassezia species but also did not affect the growth of symbiotic bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes strains. Furthermore, the inventors unexpectedly observed that fermentation extracts (cell-free supernatants) of Bacillus subtilis (such as, but not limited to, Bacillus subtilis G01, Bacillus subtilis G01_spo, and Bacillus subtilis N01) increased transepithelial electrical resistance (TEER) and increased the expression of genes involved in fatty acid synthesis, keratinization, and the synthesis of small proline-rich proteins. This indicates that the skin barrier function is improved and the skin barrier is strengthened, and therefore it is suitable for skin moisturizing, strengthening the skin barrier, improving skin barrier function, reducing dandruff, and reducing skin and scalp barriers.

[0024] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0025] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the Bacillus subtilis ferment extract is selected from the group consisting of: cell-free supernatant obtained from Bacillus subtilis ferment, whole broth ferment extract obtained from Bacillus subtilis ferment, and any combination thereof.

[0026] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin disorders, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the scalp or skin disorder is selected from the group consisting of: scalp microbiota imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, and any combination thereof.

[0027] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the composition reduces the growth of Malassezia species without affecting the growth of Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes strains.

[0028] In one embodiment, the composition is a Bacillus subtilis strain variant ferment, a Bacillus subtilis strain variant ferment extract, or a fraction of the variant ferment, wherein the Bacillus subtilis strain variant is derived from the parent Bacillus subtilis strain by knocking out the sporulation gene of the parent Bacillus subtilis strain, wherein the variant ferment, the variant ferment extract, or the fraction of the variant ferment has increased activity compared to the activity of the ferment, the ferment extract, or the fraction of the variant ferment, wherein the activity is selected from the group consisting of: reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0029] In one embodiment, the skin care product is a skin care product comprising a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0030] In one embodiment, the method is a method for providing at least one skin care benefit to a subject in need, the method comprising topically applying a skin care composition or skin care product comprising the composition to the skin or scalp of the subject, wherein the composition comprises an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof. Detailed Implementation

[0031] By reading the following detailed description, those skilled in the art will more readily understand the features and advantages of this disclosure. It should be understood that, for clarity, certain features of this disclosure described above and below in the context of individual embodiments may also be provided in combination of single elements. Conversely, for brevity, various features of this disclosure described in the context of individual embodiments may also be provided individually or in any sub-combination. It should be understood that, in the following description, embodiments related to a broad aspect of the invention are equally applicable to each of the other broad aspects of the invention related to the invention described above. It should be further understood that, unless otherwise indicated by the context, the embodiments described below may be combined.

[0032] Microorganisms, ferments and ferment extracts

[0033] As used in this article, “microorganism (or microbe)” refers to bacteria, fungi, viruses, protozoa, archaea, and other microorganisms or microscopic organisms.

[0034] In some embodiments, one or more microorganisms suitable for use in this invention may be subjected to treatments such as exposure to high temperatures, drying, gamma irradiation, or UV irradiation, thereby rendering the microorganisms non-replicating. The one or more non-replicating microorganisms suitable for use in this invention may be dead cells or living cells that have been rendered incapable of cell division. The one or more non-replicating microorganisms suitable for use in this invention may be intact cells or cells that have undergone partial or complete lysis. In some embodiments, non-replicating cells may include a mixture of intact cells and lysed cells.

[0035] One or more microorganisms suitable for use in this invention may be included in the compositions according to the invention in a live, semi-living, inactivated, or dead form. For the purposes of this invention, "inactivated" or "dead" microorganisms are those that are no longer able to form colonies in a culture. Dead or inactivated microorganisms may have intact or broken cell membranes. Dead or inactivated microorganisms can be obtained by any method known to those skilled in the art.

[0036] In one respect, the microorganisms suitable for use in this invention include Bacillus subtilis.

[0037] In some aspects, the microorganisms suitable for use in this invention include those having the 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis MDI_063_G01 (hereinafter referred to as Bacillus subtilis G01) deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149857. 1) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) Bacterial strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. 3) Bacterial strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149972. 7) Show at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.Bacterial strains with 16S ribosomal RNA sequences exhibiting 92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0038] The 16S ribosomal RNA sequence (SEQ ID NO: 1) of Bacillus subtilis G01 is as follows:

[0039]

[0040] The 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 is as follows:

[0041]

[0042] The 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 is as follows:

[0043]

[0044] The 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 is as follows:

[0045]

[0046] In one aspect of the invention, a fermentation product is provided.

[0047] As used herein, the term "fermentation product" is understood to refer to a composition (complex mixture) produced by multiplying live microorganisms (microbial strains) in a nutrient medium. Fermentation product may include cell cluster components from said microorganisms, unconsumed culture medium components, and metabolites (i.e., unused substrates and / or fermentation end products). As used herein, "cell cluster component" means any mixture of proteins, lipids (i.e., membranes), carbohydrates, extracellular polysaccharides, metabolites, etc., from multiplied microorganisms. For example, as microorganisms grow, they produce new cells that typically include additional cell clusters, such as, but not limited to, cell membranes, nucleic acids (i.e., DNA and / or RNA), internal subcellular structures, polysaccharides, and proteins (i.e., membrane-bound, secreted, and / or intracellular).

[0048] The fermentation products used in this invention include fermentation products derived from the microorganism Bacillus subtilis.

[0049] The growth medium used to prepare the ferment is any medium containing the necessary nutrients for propagating one or more microorganisms suitable for the present invention. Suitable nutrients include, but are not limited to, amino peptides, peptides, yeast extracts, salts, sugars, carbohydrates, and / or vitamins. The medium may be based on dairy products (such as milk), grains, fruits, and / or vegetables.

[0050] In one aspect, the fermentation product used in this invention comprises a fermentation product derived from the microorganism Bacillus subtilis, wherein the Bacillus subtilis is selected from the following: having a 16S ribosomal RNA sequence (SEQ ID NO: ) similar to that of Bacillus subtilis G01 with accession number CBS149857 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) Bacterial strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. 3) Bacterial strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149972. 7) Show at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.Bacterial strains with 16S ribosomal RNA sequences exhibiting 91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0051] The ferment can be further concentrated to obtain an effective amount of the active ingredient in the ferment before it is processed into a ferment extract or included in a composition. The ferment extract can be produced from the ferment as described herein. The ferment, ferment extract, and fractions thereof can be spray-dried or lyophilized before being included in a skin care composition or skin care product.

[0052] It is clear that the fermentation product can be used directly in the compositions and methods of the present invention, or one or more fractions or extracts of the fermentation product containing the active ingredient can be separated from the fermentation product by any suitable means before use.

[0053] In one aspect of the invention, a fermentation extract is provided. The fermentation extract used in the present invention comprises a fermentation extract derived from the microorganism Bacillus subtilis.

[0054] As used herein, the term "fermentation extract" refers to an extract or fraction derived from fermentation products produced by the propagation of live microorganisms (microbial strains) in a nutrient medium as described above. In one aspect, fermentation extract is an extract of fermentation products produced by propagating a species of Bacillus subtilis.

[0055] In one aspect, the ferment extract is a cell-free supernatant of the ferment. As used herein, “cell-free supernatant,” “fermentation supernatant,” “cell-free fermentation,” or “fermentation filtrate” are used interchangeably and refer to a ferment extract that is substantially free of living cells, such as the supernatant of a cell culture of at least one microorganism from which cells have been removed. It should be understood that cells can be removed from cell cultures by any method known in the art, and such cell removal (e.g., by centrifugation, filtration) will still produce a cell-free supernatant that may contain trace amounts of cells or cell debris. Methods for isolating cells from growth media are well known in the art and can rely on physical methods, such as centrifugation to produce a cell pellet and culture supernatant, filtration, ultrafiltration, tangential flow filtration, normal flow filtration, or reverse osmosis. Alternatively or additionally, the isolation method can be ligand-based and includes, for example, antibodies that specifically bind to Bacillus subtilis. Antibodies can be conjugated to a solid support, such as magnetic beads. In one embodiment, a cell-free supernatant is obtained by filtering or centrifuging a culture medium from which Bacillus subtilis cells are cultured.

[0056] On the one hand, cell-free supernatant (also known as ferment extract) is obtained by filtering or centrifuging Bacillus subtilis ferment.

[0057] On one hand, cells were removed from the Bacillus subtilis ferment by precipitating the cells (centrifuging at 4,000 to 8,000 xg) and passing the supernatant through a 0.2 μM filter to obtain a substantially cell-free supernatant.

[0058] In one aspect, the fermentation extract used in this invention is a Bacillus subtilis fermentation extract that consists essentially of cell-free fermentation. In the context of fermentation, the term "consistently of" includes at least 90% of the fermentation having the indicated properties (e.g., cell-free fermentation). Suitably, at least 95% has the indicated properties. Suitably, at least 97% has the indicated properties. Suitably, at least 99% has the indicated properties. In some embodiments, at least 100% has the indicated properties.

[0059] Fermentation extracts used in the compositions and methods and / or uses of the present invention may be substantially free of live Bacillus subtilis cells, typically containing zero (or substantially zero) live cells / mL of fermentation.

[0060] On the other hand, fermentation extract is an extract of cell pellet obtained from fermentation broth. The cell pellet can be obtained by centrifuging the fermentation broth and removing the cell-free supernatant.

[0061] On one hand, the cell precipitate extract is obtained from Bacillus subtilis fermentation broth by centrifuging the cell precipitate (4,000 to 8,000 xg) and discarding the supernatant, leaving a portion of the cell precipitate. The precipitate is resuspended in acidic water (1 / 10 v / v) to pH 2.0–4.0, vortexed, precipitated by centrifugation (4,000 to 8,000 xg), and the extract is passed through a 0.2 µM filter. Optionally, the cell precipitate can be resuspended in alkaline water (1 / 10 v / v) at a pH above 8.5. The cell precipitate can be suspended in any solution or under any conditions that produce an effective amount of the fermentation broth extract solution.

[0062] Starting with cell precipitation has the following advantages: the volume of the resuspended liquid (extract volume) can be determined to produce an effective amount of active extract and / or further concentrate it to produce an effective amount of active extract. Furthermore, resuspending the precipitate in water or a non-fermented broth liquid results in a reduction or elimination of inactive components in the fermented broth that may interfere with the activity of the cell-precipitated extract.

[0063] On the other hand, the Bacillus subtilis ferment extract is produced by combining the above-mentioned cell-free supernatant fraction with the above-mentioned cell precipitate extract.

[0064] On the other hand, Bacillus subtilis ferment extract is a whole broth ferment extract prepared by first adjusting the pH of the ferment (total ferment broth) to between pH 2.0 and 4.0, then precipitating insoluble cellular material, and optionally filtering the supernatant through a 0.2 µM filter to obtain a cell-free supernatant called "whole broth ferment extract".

[0065] Fermentation extracts may be further concentrated or purified before being included in a composition to obtain an effective amount of the fermentation extract. Fermentation extracts may be spray-dried or lyophilized before being included in a skin care composition or skin care product.

[0066] Alternatively, the Bacillus subtilis whole broth ferment extract is prepared by first adjusting the pH of the ferment (total fermented broth) to an alkaline pH, then precipitating insoluble cell material, and optionally filtering the supernatant through a 0.2 µM filter to obtain a cell-free supernatant (also known as "whole broth ferment extract").

[0067] On one hand, ferment extracts are obtained from ferments produced using nutrient media with a pH between 2 and 12.

[0068] On one hand, ferment extracts are obtained from ferments, wherein the pH of the ferments is adjusted to a pH between 2 and 12 before obtaining the ferment extracts.

[0069] It should also be understood that the production of ferments and ferment extracts can vary between batches (fermentation intervals), which can lead to different efficacies of the ferment extracts. Therefore, batch-to-batch variations in the production of effective amounts of Bacillus subtilis ferment extracts can be observed.

[0070] In one embodiment, Bacillus subtilis ferment, ferment extract, or a composition containing ferment extract is formulated in a dry or liquid preparation.

[0071] In one embodiment, Bacillus subtilis ferment, ferment extract, or a composition containing the ferment extract is formulated in at least one form selected from the group consisting of: loose or dense powder, granules, liquid suspension or solution, spray solution, or any combination thereof.

[0072] In one aspect, the particles contain approximately 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25.0%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35.0%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to 100% by weight of the Bacillus subtilis ferment extract described herein.

[0073] The particles can be produced by any means known in the art, such as, but not limited to, spray-drying the ferment extract or the ferment itself onto the core to form the particles. In one aspect, the particles are easily dispersible layered particles. The layered particles may comprise a core surrounded by a coating layer comprising at least an effective amount of Bacillus subtilis ferment extract distributed within a protectant matrix, wherein the core is water-soluble and rapidly dissolves. The protectant matrix may comprise at least one polyhydroxy compound and at least one phosphate compound.

[0074] On the one hand, Bacillus subtilis ferment, Bacillus subtilis ferment extract or fraction thereof are effective in providing at least one skin care benefit selected from the group consisting of: reducing dandruff symptoms, reducing the presence of Malassezia species on the skin or scalp, removing biofilm formation of Malassezia species on the skin or scalp, preventing or reducing biofilm formation of Malassezia species on the skin (scalp), reducing skin or scalp barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0075] Strengthening the skin barrier or improving skin barrier function means that the skin (such as, but not limited to, its permeability barrier) is "tighter / stronger" in some way, and it restricts or prevents compounds from entering (such as microorganisms, pollutants, etc.), and it also restricts or prevents the amount of water from being expelled, i.e., the skin barrier function is improved.

[0076] In one aspect, the fermentation extract or fraction thereof used in the present invention comprises a fermentation extract or fraction thereof from the microorganism Bacillus subtilis, wherein the Bacillus subtilis is selected from the group having a 16S ribosomal RNA sequence (SEQ ID NO: ) similar to that of Bacillus subtilis G01 with accession number CBS149857 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) Bacterial strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. 3) Bacterial strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149972. 7) Show at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.Bacterial strains with 16S ribosomal RNA sequences exhibiting 90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0077] As used herein, the terms “fraction of Bacillus subtilis” or “fraction of Bacillus subtilis ferment” or “fraction of Bacillus subtilis ferment extract” or “fraction thereof” more specifically refer to a fragment of Bacillus subtilis ferment or Bacillus subtilis ferment extract that is effective in providing at least one skin care benefit selected from the group consisting of: reducing dandruff symptoms, reducing the presence of Malassezia species on the skin or scalp, removing biofilm formation of Malassezia species on the skin or scalp, preventing or reducing biofilm formation of Malassezia species on the skin (scalp), reducing skin or scalp barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0078] Skin care compositions and skin care products for providing at least one skin care benefit.

[0079] As used herein, the term "skin care composition" refers to a composition containing at least one skin care beneficiary that can provide skin care benefits.

[0080] As used herein, the terms “skin care benefit agent” or “active agent” or “bioactive agent” are used interchangeably and refer to Bacillus subtilis ferment, Bacillus subtilis ferment extract or fraction thereof that can provide skin care (scalp care) benefits.

[0081] In some embodiments provided herein, the skin care beneficial agent comprises an effective amount of Bacillus ferment, Bacillus ferment extract, or fraction thereof, wherein the Bacillus is selected from the following: having a 16S ribosomal RNA sequence (SEQ ID NO: CBS149857) of Bacillus subtilis G01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) Bacterial strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, and possessing a 16S ribosomal RNA sequence (SEQ ID NO.) of Bacillus subtilis N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. NO:3) Bacterial strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO:) of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149972. 7) Show at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, and 99%.Bacterial strains with 16S ribosomal RNA sequences exhibiting 8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0082] As used herein, the term "skin care benefit" refers to the benefit provided by a skin care beneficial agent, such as a ferment, ferment extract or fraction thereof, when applied topically to the skin or scalp (or by a skin care composition and / or skin care product containing an effective amount of said skin care beneficial agent).

[0083] Surprisingly, the inventors observed that the fermentation extract (cell-free supernatant) of Bacillus subtilis (such as, but not limited to, Bacillus subtilis N01, Bacillus subtilis PC01, Bacillus subtilis G01, Bacillus subtilis G01_spo, and Bacillus subtilis K01) reduced the growth of Malassezia species. The inventors also unexpectedly observed that the fermentation extract (cell-free supernatant) of Bacillus subtilis (such as, but not limited to, Bacillus subtilis G01 and Bacillus subtilis G01_spo) not only reduced the growth of Malassezia species but also did not affect the growth of Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes strains. Furthermore, the inventors unexpectedly observed that fermentation extracts (fermentation supernatant) of Bacillus subtilis (such as, but not limited to, Bacillus subtilis G01 and Bacillus subtilis N01) increased transepithelial electrical resistance (TEER) and increased the expression of genes involved in fatty acid synthesis, keratinization, and small proline-rich protein synthesis. This indicates that the skin barrier function is improved and the skin barrier is strengthened, and therefore it is suitable for skin moisturizing, strengthening the skin barrier, improving skin barrier function, reducing dandruff, and reducing skin and scalp barriers.

[0084] In one aspect of the invention, the skin care benefits are selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin (protecting the skin from dehydration by maintaining, restoring and / or improving skin hydration), promoting skin repair, and any combination thereof.

[0085] For the purposes of this disclosure, the term "dandruff" or "dandruff syndrome" refers to a condition characterized by excessive dryness or excessive sebum production of the scalp, which, depending on the circumstances, may be characterized by the presence of dry or oily or greasy dandruff, or even itching and / or epidermal inflammation.

[0086] For the purposes of this disclosure, the term "scalp or skin disorder" includes scalp dandruff (seborrheic dermatitis), scalp microbiota imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, or any combination thereof.

[0087] Dry dandruff reflects a dry scalp, which may be combined with an excessively rapid turnover of the scalp's keratinized layer. Dry dandruff scales typically appear as small white or gray flakes and are distributed on the scalp and clothing, creating an unsightly appearance.

[0088] Itching associated with dry scalp can lead to erythema or even inflammation.

[0089] Oily or greasy scalp dandruff is a form of seborrheic dermatitis. Individuals with seborrheic dermatitis have erythematous scalp covered with numerous greasy or oily, yellowish scales that accumulate to form small bumps. They experience itchy scalp and often a burning sensation in the affected areas. These symptoms can be amplified by the presence of pathogenic microorganisms, particularly species of the genus Malassezia. These microorganisms, which release fatty acids from sebum, can impair the skin's barrier function and cause inflammation.

[0090] During dandruff, the skin barrier is imbalanced, its integrity and hydration are impaired, and its microbiome is disturbed. The scalp becomes irritated and itchy, fragile, less hydrated, and susceptible to infection.

[0091] The species of Malassezia mentioned in this article include, but are not limited to, Malassezia restricta, Malassezia globosa, Malassezia furfur (M. furfur), Malassezia sympodialis (M. sympodialis), Malassezia phylotype 5 (M. phylotype 5), other uncharacterized species of Malassezia, and any combination thereof.

[0092] Human skin is colonized by a diverse microbiome (Byrd, A., Belkaid, Y. & Segre, J. The human skin microbiome. Nat Rev Microbiol 16, 143-155; 2018). Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes (formerly known as Propionibacterium) are two dominant commensal species.

[0093] As used herein, the term "biofilm" refers to a microbial community embedded in an extracellular polymeric matrix attached to a surface. The extracellular polymeric matrix is ​​a polymeric aggregate typically composed of extracellular DNA, proteins, and polysaccharides. Biofilms may contain one or more microorganisms and further include water, and may include other trapped particles. These microorganisms may be Gram-positive or Gram-negative bacteria (aerobic or anaerobic); algae, protozoa, and / or yeast or filamentous fungi. In one embodiment, a biofilm is a living cell comprising one or more species of the genus *Malassezia*.

[0094] As used herein, “surface” means any structure with sufficient mass to allow biofilm attachment. Surfaces include hard surfaces and soft surfaces. Hard surfaces include, but are not limited to, metals, glass, ceramics, wood, minerals (rock, stone, marble, granite), aggregate materials such as concrete, plastics, composites, hard rubber materials, and plaster. Other surfaces can be biological surfaces, such as skin, scalp, or keratin.

[0095] Other beneficial agents used in skin care may include anti-dandruff actives.

[0096] Examples of anti-dandruff active agents include keratolytic agents (such as salicylic acid and various forms of sulfur), keratinization modifiers (such as zinc pyrithione, pyrithione salts, trihaloureas, triclosan, azoles, antifungal polymers, allantoin), steroids (such as topical corticosteroids, tar or keratolytic oil (coal tar), undecanoic acid, fumaric acid, allylamine and mixtures thereof, ciclopirox, octopirox, pyridoxine ethanolamine, clobetasol propionate, betamethasone valerate, tea tree oil, a mixture of thyme and camphor oil), topical antifungal agents (such as selenium sulfide, imidazoles (e.g., ketoconazole), hydroxypyridinones (e.g., ciclopirox), natural remedies (such as Malassezia species oil, aloe vera), and probiotic microorganisms. (Indian J. Dermatol, April-June 2010; 55(2): 130-134).

[0097] In one aspect, the skin care beneficial agent is selected from the group consisting of: cell-free supernatant, cell precipitate extract, and / or whole broth fermentation extract obtained from Bacillus subtilis fermentation, wherein the Bacillus subtilis is selected from the group consisting of: having a 16S ribosomal RNA sequence (SEQ ID NO: CBS149857) of Bacillus subtilis G01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. 3) Bacillus subtilis strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, and possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149972. 7) Show at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, and 99%.Bacillus subtilis strains exhibiting 16S ribosomal RNA sequence similarity of 4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possess a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0098] The skin care benefits described herein, or any effective amount of the skin care benefits described herein, may be formulated into a skin care composition.

[0099] On one hand, a skin care beneficial agent consisting of an effective amount of Bacillus subtilis ferment, Bacillus subtilis ferment extract and / or its fractions is formulated in a skin care composition.

[0100] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0101] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the strengthening of the skin barrier includes applying the composition to the subject's skin or scalp to strengthen the subject's skin barrier.

[0102] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the improvement of skin barrier function includes applying the composition to the skin or scalp of the subject to improve the skin barrier function of the subject.

[0103] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the skin moisturizing comprises applying the composition to the subject's skin or scalp to moisturize the subject's skin or scalp.

[0104] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the composition comprises an effective amount relative to the total weight of the composition of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%. 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 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%, 63%, 64 %, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% by weight of Bacillus subtilis ferment extract or fractions thereof.

[0105] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the composition comprises an effective amount relative to the total volume of the composition of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%. 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 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%, 63%, 64 %, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% by volume of Bacillus subtilis ferment extract or fractions thereof.

[0106] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the Bacillus subtilis ferment extract is selected from the group consisting of: cell-free supernatant obtained from Bacillus subtilis ferment, cell precipitate extract obtained from Bacillus subtilis ferment, whole broth ferment extract obtained from Bacillus subtilis ferment, and any combination thereof.

[0107] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin disorders, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the scalp or skin disorder is selected from the group consisting of: scalp microbiota imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, and any combination thereof.

[0108] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the reduction of dandruff comprises applying the composition to the skin or scalp of the subject to reduce dandruff.

[0109] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the Bacillus subtilis ferment extract or a fraction thereof is obtained from the fermentation of Bacillus subtilis selected from the group consisting of: having the 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01 with accession number CBS149857 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. 3) Show at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.Bacillus subtilis strains with 99% to at most 100% sequence similarity to the 16S ribosomal RNA sequence of Bacillus subtilis K01, deposited at the Westdick Institute for Fungal Biodiversity (WFDB), have the same 16S ribosomal RNA sequence (SEQ ID NO: 7) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS151098. 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0110] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, the composition further comprising one or more anti-dandruff active agents.

[0111] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the composition reduces the growth of Malassezia species without affecting the growth of Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes strains.

[0112] In one embodiment, a skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reduction of dandruff, reduction of scalp or skin barriers, strengthening of the skin barrier, improvement of skin barrier function, skin hydration, and any combination thereof, wherein the strengthening of the skin barrier and / or the improvement of skin barrier function and / or the skin hydration occurs through fatty acid synthesis, keratinization, synthesis of small proline-rich proteins, and any combination thereof.

[0113] On the one hand, fatty acid synthesis or increased fatty acid synthesis (when compared with the same skin care composition lacking the effective amount of Bacillus subtilis ferment extract or its fractions) occurs through fatty acid extension.

[0114] On one hand, fatty acid synthesis or increased fatty acid synthesis (when compared with the same skin care composition lacking the effective amount of Bacillus subtilis ferment extract or its fraction) occurs through fatty acid elongation, wherein the elongation of said fatty acids occurs through increased expression of the very long chain fatty acid elongase family (ELOVL) fatty acid elongase 7 (ELVOL7) (when compared with the same skin care composition lacking the effective amount of Bacillus subtilis ferment extract or its fraction).

[0115] On the one hand, keratinization or increased keratinization (when compared to the same skin care composition lacking the effective amount of Bacillus subtilis ferment extract or fraction thereof) occurs through increased expression of late keratinization capsule 1c (LCE1C) protein (when compared to the same skin care composition lacking the effective amount of Bacillus subtilis ferment extract or fraction thereof).

[0116] On the one hand, the synthesis of small proline-rich proteins or the increase in small proline-rich protein synthesis (when compared with the same skin care composition lacking the effective amount of the Bacillus subtilis ferment extract or its fraction) occurs through the increased expression of small proline-rich proteins (SPRR) (such as, but not limited to, SPRR selected from the group consisting of SPRR2F, SPRR2E, SPRR2A, SPRR2D, and any combination thereof).

[0117] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa.

[0118] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa by weight, between 0.1% and 100% of the total weight of the effective amount.

[0119] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa by volume, between 0.1% and 100% of the total volume relative to the effective amount.

[0120] In one embodiment, the skin care composition is a skin care composition as described herein, wherein the at least one active agent is bacitracin.

[0121] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reduction of dandruff, reduction of scalp or skin disorders, Malassezia growth inhibition, reduction of Malassezia species growth without affecting the growth of Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes strains, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa.

[0122] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reduction of dandruff, reduction of scalp or skin disorders, Malassezia growth inhibition, reduction of Malassezia species growth without affecting the growth of Staphylococcus aureus and Propionibacterium acnes strains, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa by volume, from 0.1% to 100% of the total volume relative to the effective amount.

[0123] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reduction of dandruff, reduction of scalp or skin disorders, Malassezia growth inhibition, reduction of Malassezia species growth without affecting the growth of Staphylococcus aureus and Propionibacterium acnes strains, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa by weight, between 0.1% and 100% of the total weight relative to the effective amount.

[0124] In one embodiment, the skin care composition is the skin care composition described herein, wherein the at least one active agent is bacitracin.

[0125] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: skin barrier strengthening, improved skin barrier function, skin moisturizing, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent selected from the group consisting of: an active agent of less than 100 Kda, an active agent of less than 50 Kda, an active agent of less than 30 Kda, an active agent of less than 10 Kda, an active agent of less than 3 Kda, and any combination thereof.

[0126] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises an active agent of less than 3 kDa.

[0127] It should be further understood that, in addition to active agents derived from Bacillus subtilis (such as Bacillus subtilis ferment, Bacillus subtilis ferment extract and / or Bacillus subtilis ferment fraction), the skin care compositions used in the present invention may further comprise one or more probiotic bacteria.

[0128] Skin care compositions may contain additional compounds selected from the group consisting of: fragrances, excipients, preservatives, pH adjusters, aqueous carriers, alcohol carriers, emollients, curing agents, hydrating agents, emulsifiers, solubilizers, surfactants, thickeners, salts, and any combination thereof.

[0129] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, the composition further comprising additional compounds selected from the group consisting of: fragrances, excipients, preservatives, pH adjusters, aqueous carriers, alcohol carriers, emollients, curing agents, hydrating agents, emulsifiers, solubilizers, surfactants, thickeners, salts, and any combination thereof.

[0130] Preservatives include, but are not limited to, parabens, sodium benzoate, potassium sorbate, phenylethanol, ethyl lauroyl arginine (LAE), and any combination thereof.

[0131] pH adjusters include, but are not limited to, weak acids, strong acids, any compound that can adjust pH (such as, but not limited to, citric acid), or any combination thereof.

[0132] In one embodiment, the composition is a Bacillus subtilis strain variant ferment, a Bacillus subtilis strain variant ferment extract, or a fraction of the variant ferment, wherein the Bacillus subtilis strain variant is derived from the parent Bacillus subtilis strain by knocking out at least one sporulation gene of the parent Bacillus subtilis strain, wherein the variant ferment, the variant ferment extract, or the fraction of the variant ferment has increased activity compared to the activity of a ferment, a ferment extract, or a fraction of the variant ferment derived from the parent Bacillus subtilis strain, wherein the activity is selected from the group consisting of: reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0133] In one embodiment, the composition is a Bacillus subtilis strain variant ferment, a Bacillus subtilis strain variant ferment extract, or a fraction of the variant ferment, wherein the Bacillus subtilis strain variant is derived from the parent Bacillus subtilis strain by knocking out the SpoIIE sporulation gene of the parent Bacillus subtilis strain, wherein the variant ferment, the variant ferment extract, or the fraction of the variant ferment has increased activity compared to the activity of a ferment, a ferment extract, or a fraction of the variant ferment derived from the parent Bacillus subtilis strain, wherein the activity is selected from the group consisting of: reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0134] The skin care compositions described herein can be used in formulations and skin care products.

[0135] As used herein, “skin care product” means a product containing the skin care composition described herein, including but not limited to cosmetic products, aqueous solutions, lotions, serums, gels, patches, lotions, topical moisturizers, creams, pastes, balms, ointments, hair oils, gels, liquids, sprays, shampoos, foams, kits, or any combination thereof.

[0136] In one embodiment, the skin care product is a skin care product comprising the skin care composition described herein.

[0137] In one embodiment, a skin care product is a skin care product comprising the skin care composition described herein and one or more dermatologically or skin care-acceptable components.

[0138] In one embodiment, the skin care product is a skin care product comprising a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0139] In one embodiment, a skin care product is a product comprising a skin care composition for use in reducing skin or scalp disorders, the composition comprising an effective amount of Bacillus subtilis ferment, Bacillus subtilis ferment extract and / or fractions thereof, and one or more dermatologically or skin care-acceptable components, wherein the composition reduces and / or treats the skin or scalp disorder.

[0140] In one embodiment, a skin care product is a skin care product comprising a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein Bacillus subtilis is selected from the group consisting of: having a 16S ribosomal RNA sequence (SEQ ID NO: ) similar to that of Bacillus subtilis G01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with serial number CBS149857. 1) Bacillus subtilis exhibiting a 16S ribosomal RNA sequence with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, and possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) Bacillus subtilis exhibiting a 16S ribosomal RNA sequence with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity. Bacillus subtilis suitable for this invention also includes: a 16S ribosomal RNA sequence (SEQ ID NO: ) having the same sequence as Bacillus subtilis N01 with accession number CBS149971 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 3) Bacillus subtilis exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to the 16S ribosomal RNA sequence of Bacillus subtilis K01 (SEQ ID NO: 7) deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with number CBS149972 exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.Bacillus subtilis with 16S ribosomal RNA sequences exhibiting 5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possesses a 16S ribosomal RNA sequence (SEQ ID NO: 5%) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0141] In one embodiment, the skin care product comprises at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% of the skin care composition described herein, relative to the total weight of the skin care product.

[0142] In one embodiment, the skin care product comprises at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% of the skin care composition described herein by volume relative to the total volume of the skin care product.

[0143] In one embodiment, the skin care product described herein is formulated for topical application.

[0144] In one embodiment, the skin care product described herein is formulated for topical application to the skin or scalp.

[0145] In one embodiment, a skin care product is a product comprising a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, wherein the skin care product is selected from the group consisting of: lotions, serums, gels, creams, gels, hydrogels, lotions, solid cosmetics, films, patches, shampoos, loose or dense powders, liquid suspensions or solutions, spray solutions and sticks, and any combination thereof.

[0146] In one embodiment, the skin care product is selected from the group consisting of: lotions, serums, gels, creams, gels, hydrogels, lotions, solid cosmetics, films, patches, shampoos, loose or dense powders, liquid suspensions or solutions, spray solutions, and sticks.

[0147] In one embodiment, the skin care product is a lotion, serum, gel, cream, gel, hydrogel, lotion, solid cosmetic, film, patch, shampoo, loose or dense powder, liquid suspension or solution, spray solution or stick, the skin care product comprising one or more dermatologically or skin care acceptable components and at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% by weight of the skin care composition described herein relative to the total weight of the skin care formulation.

[0148] In one embodiment, the skin care product is a product comprising a first skin care composition and a second skin care composition, wherein the first skin care composition comprises an effective amount of Bacillus subtilis ferment and / or its fractions, and wherein the second skin care composition comprises at least an effective amount of at least one second active agent selected from anti-dandruff active agents for topical application. Examples of such anti-dandruff agents include keratolytic agents (such as salicylic acid and various forms of sulfur), keratinization modifiers (such as zinc pyrithione, pyrithione salts, trihaloureas, triclosan, azoles, antifungal polymers, allantoin), steroids (such as topical corticosteroids, tar or keratolytic oil (coal tar), undecanoic acid, fumaric acid, allylamine and mixtures thereof, ciclopirox, octopirox, pyridoxine ethanolamine, clobetasol propionate, betamethasone valerate, tea tree oil, a mixture of thyme and camphor oil), topical antifungal agents (such as selenium sulfide, imidazoles (e.g., ketoconazole), hydroxypyridinones (e.g., ciclopirox), natural remedies (such as Malassezia species oil, aloe vera), and probiotic microorganisms. (Indian J. Dermatol, April-June 2010; 55(2): 130-134).

[0149] In one embodiment, the skin care product is a product comprising a first skin care composition and a second skin care composition, wherein the first skin care composition comprises an effective amount of Bacillus subtilis ferment and / or fractions thereof; wherein the second skin care composition comprises at least an effective amount of at least one second active agent selected from anti-dandruff active agents for topical application; wherein the first skin care composition is formulated into at least one form selected from the group consisting of: gel, lotion, hydrogel, loose or dense powder, liquid suspension or solution, or spray solution.

[0150] Topical formulations used in this invention can be in any form suitable for application to the scalp or skin surface, such as creams, lotions, sprays, solutions, gels, ointments, pastes, plasters, coatings, bioadhesives, suspensions, etc., and / or can be prepared to contain liposomes, micelles, and / or microspheres. Such formulations can be used in combination with occlusive coverings so that moisture evaporating from the skin surface during and after application is retained within the formulation.

[0151] Topical formulations include those in which one or more active ingredients are dissolved or dispersed in dermatological media known in the art (e.g., aqueous or non-aqueous gels, ointments, water-in-oil or oil-in-water emulsions). Such media may contain water, aqueous buffer solutions, non-aqueous solvents (e.g., ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycogen, or glycerin), oils (e.g., mineral oils such as liquid paraffin, natural or synthetic triglycerides), or silicone oils such as dimethicone). Depending on the nature of the formulation and its intended use and application site, the dermatological mediator used may contain one or more components selected from the following list (e.g., components other than water when the formulation is an aqueous gel): solubilizers or solvents (e.g., β-cyclodextrin (such as hydroxypropyl β-cyclodextrin), or alcohols or polyols (such as ethanol, propylene glycol, or glycerin)); thickeners (e.g., hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, or carbomer); gelling agents (e.g., polyoxyethylene-polyoxypropylene copolymers); preservatives (e.g., benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorobutanol, benzoates, potassium sorbate, or EDTA or salts thereof); and one or more pH buffers (e.g., a mixture of dihydrogen phosphate and hydrogen phosphate, or a mixture of citric acid and hydrogen phosphate).

[0152] Skin care products include liquid lotions (true solutions) that contain water as a solvent and water-soluble additives (solutes), such as, but not limited to, active ingredients, fragrances, colorants, preservatives, pH adjusters, chelating agents, or any combination thereof.

[0153] Skin care products include dispersions such as emulsions (including, but not limited to, liquid-in-liquid [water-in-oil W / O, O / W, W / O / W], suspensions [solid / liquid or liquid / solid], aerosols [liquid / gas or solid / gas], foams / mousses [gas / liquid or gas / emulsion, or gas / solid]). Examples of oil-in-water [O / W] emulsions include, but are not limited to, combinations of an aqueous phase, emulsifiers, fatty phases, and at least one additive. The aqueous phase may contain water, wetting agents, and stabilizers (including, but not limited to, synthetic polymers, carbomer, natural polymers, xanthan gum, gum arabic, carrageenan, gellan gum, or any combination thereof). Emulsifiers include, but are not limited to, anionic emulsifiers, cationic emulsifiers, nonionic emulsifiers, amphoteric emulsifiers, silicone emulsifiers, and auto-emulsifiers. Fatty phases (lipophilic components) include, but are not limited to, waxes, butter, fatty esters, triglycerides, vegetable oils, mineral oils (paraffin), silicones, and thickeners / oil gelling agents. Additives include, but are not limited to, preservatives, fragrances (most commonly lipophilic), colorants, antioxidants, chelating agents, active ingredients, pH adjusters (citric acid, lactic acid, AHA), neutralizers / strong alkaline reagents (such as NaOH, trimethylamine (used for gelling acrylic polymers)), and powders.

[0154] Skin care products include aqueous gels, which contain an aqueous phase (including water, wetting agents, and active ingredients), gelling agents (such as, but not limited to, synthetic polymers, natural polymers, xanthan gum, gum arabic, carrageenan, and gellan gum), and additives (such as, but not limited to, fragrances, high-HLB surfactants, colorants, active ingredients, preservative systems, pH adjusters, neutralizers, and powders).

[0155] Skin care products include cleansing / surfactant systems (such as, but not limited to, shampoos, shower gels, micellar water), which include an aqueous phase (water, wetting agent), surfactants, additives (such as, but not limited to, fragrances, high HLB surfactants, colorants, active ingredients, preservative systems, pH adjusters, neutralizers, powders), and optionally gelling agents (such as, but not limited to, synthetic polymers, natural polymers, xanthan gum, gum arabic, carrageenan, gellan gum).

[0156] Dermatologically or skincare-acceptable carriers can also be incorporated into the skincare products (formulations) of the present invention, and these can be any carrier conventionally used in the art. Examples include water, lower alcohols, higher alcohols, polyols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water-based and emulsion-based mixtures of such carriers.

[0157] The terms “dermatologically acceptable” or “dermatologically acceptable carrier” or “skincare acceptable” or “skincare acceptable carrier” are used herein to refer to compounds or compositions that can be incorporated into dermatological or skincare formulations without causing undesirable biological effects or unwanted interactions with other components of the formulation.

[0158] As used herein, “carrier” or “medium” refers to a carrier material suitable for incorporation into a composition for topical application. Carriers and mediators that may be used herein include any such materials known in the art that are nontoxic and do not interact in a harmful manner with other components of a formulation comprising them.

[0159] The term "aqueous" refers to a formulation that contains water or becomes water-containing after being applied to skin or mucous membrane tissue.

[0160] The skin care products described herein may further comprise one or more dermatologically or skin care-acceptable components known or otherwise effectively used in skin care, provided that these optional components are physically and chemically compatible with the essential components described herein, or do not otherwise unduly impair the product's stability, aesthetics, or performance. Non-limiting examples of such optional components are disclosed in the International Skin Care Ingredient Dictionary, 9th Edition, 2002 and the CTFA Skin Care Ingredient Handbook, 10th Edition, 2004.

[0161] On one hand, a dermatologically or skincare-acceptable component is a dermatologically acceptable carrier comprising about 10 wt.% to about 99.9 wt.%, alternatively about 50 wt.% to about 95 wt.%, and alternatively about 75 wt.% to about 95 wt.%. Carriers suitable for use with one or more compositions may include, for example, those used in formulations of mousses, tonics, gels, skin moisturizers, and lotions. Carriers may comprise water; organic oils; silicones, such as volatile silicones, amino or non-amino silicone gums or oils and mixtures thereof; mineral oils; vegetable oils, such as olive oil, castor oil, rapeseed oil, coconut oil, wheat germ oil, sweet almond oil, avocado oil, macadamia nut oil, apricot oil, safflower oil, candelilla oil, linseed oil, tamanu oil, lemon oil, and mixtures thereof; waxes; and organic compounds, such as C2-C... 10 Alkanes, acetone, methyl ethyl ketone, volatile organic compounds (C1-C) 12 alcohols, C1-C 20Esters of acids and C1-C8 alcohols (such as methyl acetate, butyl acetate, ethyl acetate, and isopropyl myristate), dimethoxyethane, diethoxyethane, C 10 -C 30 Fatty alcohols (such as lauryl alcohol, cetyl alcohol, stearyl alcohol, and behenyl alcohol); C 10 -C 30 Fatty acids, such as lauric acid and stearic acid; C 10 -C 30 Fatty amides, such as lauric acid diethanolamide; C 10 -C 30 Fatty alkyl esters, such as C 10 -C 30 Fatty alkyl benzoates; hydroxypropyl cellulose; and mixtures thereof. In one aspect, the carrier comprises water, fatty alcohols, volatile organic alcohols, and mixtures thereof. Other carriers can be formulated by those skilled in the art.

[0162] The skin care products described herein may further include from about 0.1% to about 10% and alternatively from about 0.2% to about 5.0% of a gelling agent to help provide the desired viscosity to one or more compositions. Non-limiting examples of suitable optional gelling agents include crosslinked carboxylic acid polymers; unneutralized crosslinked carboxylic acid polymers; unneutralized modified crosslinked carboxylic acid polymers; crosslinked ethylene / maleic anhydride copolymers; unneutralized crosslinked ethylene / maleic anhydride copolymers (e.g., EMA 81, commercially available from Monsanto); unneutralized crosslinked alkyl ether / acrylate copolymers (e.g., SALCARE™ SC90, commercially available from Allied Colloids); unneutralized crosslinked copolymers of sodium polyacrylate, mineral oil, and PEG-1 tridecyl ether-6 (e.g., SALCARE™ SC91, commercially available from Allied Colloids); unneutralized crosslinked copolymers of methyl vinyl ether and maleic anhydride (e.g., STABILEZE™ QM-PVM / MA copolymer, commercially available from International Specialty Products); hydrophobically modified nonionic cellulose polymers; hydrophobically modified ethoxylated urethane polymers (e.g., from Union Carbide... (Carbide) commercially available UCARE™ Polyphobe series alkali-swellable polymers; and combinations thereof. In this context, the term "unneutralized" means that the optional polymer and copolymer gelling agent materials contain unneutralized acid monomers.

[0163] Dermatologically or skincare-acceptable media may contain fatty substances, typically in proportions of about 10% to about 90% by weight relative to the total weight of the product, wherein the fatty phase contains at least one liquid, solid, or semi-solid fatty substance. Fatty substances include, but are not limited to, oils, waxes, gums, and so-called paste-like fatty substances. Alternatively, the product may be in the form of a stable dispersion, such as a water-in-oil or oil-in-water emulsion. Additionally, skincare products may contain one or more conventional skincare or dermatological additives or adjuvants, including but not limited to antioxidants, preservatives, fillers, surfactants, UVA and / or UVB sunscreens, fragrances, thickeners, wetting agents, and anionic, nonionic, or amphoteric polymers, as well as dyes or pigments (colorants).

[0164] A dermatologically acceptable carrier may be a moisturizing formulation containing at least one emulsifier, at least one surfactant, or any combination thereof.

[0165] Skin care compositions and skin care products may further comprise skin care active ingredient materials, including sunscreens, moisturizers, humectants, skin-beneficial agents, depositing agents such as surfactants, occlusive agents, moisture barriers, lubricants, emollients, anti-aging agents, antistatic agents, abrasives, antimicrobial agents, conditioning agents, exfoliating agents, fragrances, thickeners, salts, lipids, phospholipids, vitamins, foam stabilizers, pH adjusters, preservatives, suspending agents, silicone oils, silicone derivatives, essential oils, oils, fats, fatty acids, fatty acid esters, fatty alcohols, waxes, polyols, hydrocarbons, and mixtures thereof.

[0166] Other ingredients that may be included in a skin care composition or skin care product include, but are not limited to, at least one active ingredient (such as zinc oxide, petrolatum, white petrolatum, mineral oil, cod liver oil, lanolin, dimethicone, stearin, vitamin A, allantoin, calamine, kaolin, glycerin, or colloidal oatmeal, and combinations thereof) for reducing or preventing skin conditions, providing skin care effects, or providing moisturizing benefits to the skin, and one or more natural moisturizing factors (such as ceramides, hyaluronic acid, etc.). Oils, squalane, amino acids, cholesterol, fatty acids, triglycerides, phospholipids, glycosphingolipids, urea, linoleic acid, glycosaminoglycans, mucopolysaccharides, sodium lactate or sodium pyrrolidone carboxylate), glycerides, almond oil, low erucic acid rapeseed oil, squalane, squalene, coconut oil, corn oil, jojoba oil, jojoba wax, lecithin, olive oil, safflower oil, sesame oil, shea butter, soybean oil, sweet almond oil, sunflower oil, tea tree oil, shea butter, palm oil, cholesterol, cholesterol esters, wax esters, fatty acids, and orange oil.

[0167] Any number of dermatologically acceptable materials commonly used in skin care products can also be incorporated into the skin care products of the present invention (such as skin conditioning agents and skin coloring agents).

[0168] Skin conditioning agents, as defined herein, include, but are not limited to, astringents that tighten the skin; exfoliating agents that remove dead skin cells; emollients that help maintain a smooth, soft, and supple appearance; humectants that increase the moisture content of the outer layer of the skin; occlusives that delay the evaporation of water from the skin surface; and various compounds that enhance the appearance of dry or damaged skin or reduce peeling and restore softness. Skin conditioning agents are well known in the art, see, for example, Green et al. (WO 01 / 07009), and are commercially available from various sources. Suitable examples of skin conditioning agents include, but are not limited to, lactobionic acid, gluconic acid, α-hydroxy acids, β-hydroxy acids, polyols, hyaluronic acid, D,L-panthenol, polysalicylic acid esters, vitamin A palmitate, vitamin E acetate, glycerin, sorbitol, silicones, silicone derivatives, lanolin, natural oils, xylitol, fucose, rhamnose, xylitol, betaine, and triglycerides. Skin conditioning agents may include polysalicylic acid esters, propylene glycol (CAS No. 57-55-6, Dow Chemical, Midland, MI), glycerin (CAS No. 56-81-5, Proctor & Gamble Co., Cincinnati, OH), glycolic acid (CAS No. 79-14-1, DuPont Co., Wilmington, DE), and lactic acid (CAS No. 50-21-5, Alfa Aesar, Ward Hill, Massachusetts). Polysalicylic acid esters can be prepared by the method described by White et al. in U.S. Patent No. 4,855,483, which is incorporated herein by reference. Gluconic acid esters can be synthesized by the method described by Merbouh et al. (Carbohydr. Res. [Carbohydrate Research] 336:75-78 (2001)). 3-Hydroxyvalerate can be prepared as described by Bramucci in his published international patent application number WO 02 / 012530.

[0169] Skin care compositions and skin care products may contain skin care additives, such as, but not limited to, colorants / dyes, fragrances, active ingredients, preservatives, pH adjusters, chelating agents, and antioxidants.

[0170] The skin care compositions and skin care products described herein may also be part of a kit for providing one or more skin care benefits (such as, but not limited to, kits for reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, or any combination thereof).

[0171] In one aspect, the kit is a kit containing a skin care product for reducing dandruff in a subject in need, and written instructions for applying the skin care product to the subject in need, wherein the skin care product contains an effective amount of the Bacillus subtilis ferment extract and / or fractions thereof described herein.

[0172] Methods for providing at least one skin care benefit to the skin or scalp

[0173] The skin care compositions and skin care products described herein can be used in methods for providing at least one skin care benefit to subjects in need.

[0174] In one respect, the skin care compositions and skin care products described herein may be used in methods for providing at least one skin care benefit to a subject in need, wherein the skin care benefit is selected from the group consisting of: reducing dandruff, reducing the presence of Malassezia species on the skin or scalp, removing biofilms of Malassezia species formed on the skin or scalp, preventing or reducing the formation of Malassezia species biofilms on the skin (scalp), reducing skin or scalp barriers, strengthening the skin barrier, improving skin barrier function, skin moisturizing (protecting the skin from dehydration by maintaining, restoring and / or strengthening skin hydration), and any combination thereof.

[0175] In one embodiment, the method is a method for providing at least one skin care benefit to a subject in need, the method comprising topically applying a skin care composition or skin care product comprising the composition to the skin or scalp of the subject, wherein the composition comprises an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing the presence of Malassezia species on the skin or scalp, removing biofilm formation of Malassezia species on the skin or scalp, preventing or reducing biofilm formation on the skin (scalp), reducing skin or scalp barriers, strengthening the skin barrier, improving skin barrier function, skin moisturizing (protecting the skin from dehydration by maintaining, restoring and / or strengthening skin hydration), and any combination thereof. In one aspect, the Bacillus subtilis ferment extract is selected from the group consisting of: cell-free supernatant obtained from Bacillus subtilis ferment, cell precipitate extract obtained from Bacillus subtilis ferment, whole broth ferment extract obtained from Bacillus subtilis ferment, and any combination thereof.

[0176] In one embodiment, the method is a method for reducing dandruff in a subject, the method comprising applying topically to the subject's scalp a skin care composition or skin care product containing the skin care composition, wherein the skin care composition contains an effective amount of Bacillus subtilis ferment extract or a fraction thereof.

[0177] In one embodiment, the method is a method for reducing scalp or skin disorders in a subject, the method comprising topically applying a skin care composition or skin care product comprising the skin care composition to the scalp of the subject, wherein the skin care composition comprises an effective amount of Bacillus subtilis ferment extract or a fraction thereof. In one aspect, the scalp or skin disorder is selected from the group consisting of: scalp dandruff (seborrheic dermatitis), scalp microbiota imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, and any combination thereof.

[0178] In one embodiment, the method is a method for strengthening the skin barrier of a subject, the method comprising applying the skin care composition or skin care product described herein topically to the skin or scalp of the subject.

[0179] In one embodiment, the method is a method for moisturizing the skin of a subject, comprising applying the skin care composition or skin care product described herein topically to the skin or scalp of the subject.

[0180] In one aspect, the Bacillus subtilis ferment, Bacillus subtilis ferment extract, or fraction thereof used in the methods described herein are Bacillus subtilis ferments selected from the group consisting of: ferments containing the 16S ribosomal RNA sequence (SEQ ID NO: CBS149857) of Bacillus subtilis G01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. 3) Bacillus subtilis strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, and possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149972. 7) Show at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, and 99.Bacillus subtilis strains exhibiting 16S ribosomal RNA sequence similarity of 6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possess a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0181] It is further evident that the skin care composition used according to the present invention may contain, for example, at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 3% by volume relative to the total volume of the skin care composition. 7%, 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%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% of Bacillus subtilis ferment, Bacillus subtilis ferment extract and / or fractions thereof.

[0182] In one aspect, the skin care composition used according to the present invention may contain, for example, at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37% of the total weight of the skin care composition, by weight. %, 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%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% Bacillus subtilis ferment, Bacillus subtilis ferment extract and / or its fractions.

[0183] On one hand, a skin care product is applied to subjects in need, wherein the skin care product comprises at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% by weight relative to the total weight of the skin care product. 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, up to 50% of the skin care composition described herein.

[0184] In one aspect, the skin care compositions or skin care products used according to the present invention are selected from the group consisting of: lotions, serums, gels, creams, gels, lotions, solid cosmetics, films, patches, shampoos and sticks.

[0185] Methods for increasing the activity of Bacillus subtilis strain fermentation extracts and their compositions

[0186] This article discloses a method for increasing the activity of Bacillus subtilis strain ferment extracts used to provide at least one skin care benefit.

[0187] The inventors unexpectedly observed that when a sporulation gene (such as, but not limited to, SpoIIE) in a parental Bacillus subtilis strain (such as, but not limited to, Bacillus subtilis E04) was knocked out to produce a Bacillus subtilis variant strain (such as, but not limited to, Bacillus subtilis E04_spo), the activity of the ferment, fermentation extract, or fraction derived from the parental Bacillus subtilis strain was increased compared to the activity of the ferment, fermentation extract, or fraction thereof, wherein the activity is selected from the group consisting of: reducing dandruff, reducing the presence of Malassezia species on the skin or scalp, strengthening the skin barrier, improving skin barrier function, and any combination thereof.

[0188] In one embodiment, the method is a method for increasing the activity of a Bacillus subtilis strain ferment extract for providing at least one skin care benefit, the method comprising: (a) providing a parent Bacillus subtilis strain capable of producing an effective amount of the ferment extract for providing at least one skin care benefit selected from the group consisting of: reduced dandruff, reduced presence of Malassezia species on the skin or scalp, reduced scalp or skin barriers, strengthened skin barrier, improved skin barrier function, skin hydration, and any combination thereof; (b) modifying the parent Bacillus subtilis strain by knocking out at least one sporulation gene from the genome of the Bacillus subtilis strain, thereby producing a variant strain that cannot form sporulations; and (c) A fermentation extract of the Bacillus subtilis variant strain, wherein the fermentation extract of the Bacillus subtilis variant strain has increased activity when compared with the activity of the fermentation extract of the parent Bacillus subtilis strain, is intended to provide in subjects of need at least one skin care benefit selected from the group consisting of: reduced dandruff, reduced presence of Malassezia species on the skin or scalp, reduced scalp or skin barriers, strengthened skin barrier, improved skin barrier function, skin hydration, and any combination thereof.

[0189] In one embodiment, the method is a method for increasing the activity of a Bacillus subtilis strain ferment extract for providing at least one skin care benefit, the method comprising: (a) providing a parent Bacillus subtilis strain capable of producing an effective amount of the ferment extract for providing at least one skin care benefit selected from the group consisting of: reduced dandruff, reduced presence of Malassezia species on the skin or scalp, reduced scalp or skin barriers, strengthened skin barrier, improved skin barrier function, skin hydration, and any combination thereof; (b) modifying the parent Bacillus subtilis strain by knocking out the SpoIIE sporulation gene from the genome of the Bacillus subtilis strain, thereby producing a variant strain that cannot form sporulations; and (c) A fermentation extract of the Bacillus subtilis variant strain, wherein the fermentation extract of the Bacillus subtilis variant strain has increased activity when compared with the activity of the fermentation extract of the parent Bacillus subtilis strain, is intended to provide in subjects of need at least one skin care benefit selected from the group consisting of: reduced dandruff, reduced presence of Malassezia species on the skin or scalp, reduced scalp or skin barriers, strengthened skin barrier, improved skin barrier function, skin hydration, and any combination thereof.

[0190] General definition

[0191] All cited patent and non-patent literature disclosures are incorporated into this paper in their full text by reference.

[0192] Many terms and abbreviations are used in this disclosure. Unless otherwise specified, the following definitions apply.

[0193] As used herein, the articles “a / an” and “the” preceding an element or component of the invention are intended to be non-limiting in terms of the number of instances (i.e., occurrences) of that element or component. Therefore, “a / an” and “the” should be understood to include one or at least one, and the singular form of an element or component also includes the plural unless the number clearly indicates a singularity.

[0194] When quantities, concentrations, or other values ​​or parameters are given as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether these ranges are disclosed individually. When numerical ranges are listed herein, unless otherwise stated, the range is intended to include its endpoints, as well as all integers and fractions within that range. Ranges are not intended to be limited to the specific values ​​listed when defining a range.

[0195] Unless otherwise expressly indicated, the use of numerical values ​​within the ranges specified in this application is stated as approximations, as the minimum and maximum values ​​within the stated ranges are preceded by the word "about". In this way, slightly higher and lower than the stated ranges can achieve substantially the same results as values ​​within these ranges. Moreover, the disclosure of these ranges is intended as a continuous range including each value between the minimum and maximum. As used herein, the term "about" modifying the amount of an ingredient or reactant used refers to a quantitative variation that may occur, for example, by: typical measurement and liquid handling procedures used in the preparation of concentrates or the use of solutions in the real world; unintentional errors in these procedures; differences in the manufacture, source, or purity of the ingredients used to prepare the composition or carry out the method; etc. The term "about" also covers amounts that differ due to different equilibrium conditions resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalent amounts of these quantities.

[0196] As used herein, “administer (or administering)” means the act of introducing one or more microorganisms (microbial strains), ferments, ferment extracts or fractions thereof, one or more skin care compositions, one or more skin care formulations and / or one or more skin care products to a subject in need to provide at least one skin care benefit, such as, but not limited to, reduction of dandruff, reduction of scalp or skin barriers, strengthening of the skin barrier, improvement of skin barrier function, skin hydration, and any combination thereof.

[0197] Applying one or more microorganisms (microbial strains), ferments, ferment extracts or fractions thereof, one or more skin care compositions, one or more skin care formulations and / or one or more skin care products to a subject includes applying or introducing one or more microorganisms (microbial strains), ferments, ferment extracts or fractions thereof, one or more skin care compositions, one or more skin care formulations and / or one or more skin care products to the scalp, skin surface and in vitro or in vivo skin cells.

[0198] As used herein, the term “biocontaminant” refers to one or more unwanted and / or pathogenic biological entities, including but not limited to microorganisms, spores, viruses, prions, and mixtures thereof.

[0199] As used herein, the term "comprising" means the presence of a feature, integer, step, or component as described in the claims, without excluding the presence or addition of one or more other features, integers, steps, components, or groups thereof. The term "comprising" is intended to include embodiments covered by the terms "substantially constitutes" and "consistent with". Similarly, the term "substantially constitutes" is intended to include embodiments covered by the term "consistent with".

[0200] As used herein, the terms “embodiment” or “disclosure” are not intended to be limiting, but are generally applicable to any embodiment defined in the claims or described herein. These terms are used interchangeably herein.

[0201] As used herein, the term "excipient" refers to an inactive substance in a formulation that serves as a carrier for the active ingredient. Excipients can be used to stabilize the active ingredient in a formulation, such as for its storage stability. Excipients are sometimes also used to expand the scope of formulations containing active ingredients. "Active ingredient" includes skin care benefits as described herein.

[0202] As used herein, the term "effective amount" refers to an amount sufficient to achieve the desired effect. The desired effect includes, but is not limited to, reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0203] As used in this article, “preventing” and its grammatical variations refer to methods of partially or completely delaying or preventing the occurrence or recurrence of one or more of a disorder or condition (such as scalp disorder, dry skin, skin irritation) and / or its accompanying symptoms, or preventing a subject from acquiring or re-acquiring a disorder or condition or reducing the risk of a subject acquiring or re-acquiring one or more of a disorder or condition or its accompanying symptoms.

[0204] As used herein, the term “reducing” and its grammatical variations, relating to a particular trait, characteristic, feature, biological process, or phenomenon, refers to a reduction in that trait, characteristic, feature, biological process, or phenomenon. A trait, characteristic, feature, biological process, or phenomenon may be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%.

[0205] The terms “percent by weight (wt.%)” and “weight-to-weight percentage (% w / w)” are used interchangeably herein. Weight percentage refers to the percentage of a material by its mass when it is contained in a composition, mixture, solution, or product.

[0206] The terms “percent by volume / volume percentage (v%)” and “volume-volume percentage (% v / v)” are used interchangeably herein. Volume percentage refers to the percentage of a material by volume when it is contained in a composition, mixture, solution, or product.

[0207] The term "16S rRNA" or "16S ribosomal RNA" refers to the rRNA that makes up the small subunit of the ribosome in prokaryotes. In bacteria, this sequence can be used to identify and characterize operational taxonomic units.

[0208] The term "ITS" or "internal transcribed spacer" refers to the regions of ribosomal transcripts that are excised and degraded during maturation. Their sequences can be used for phylogenetic analysis and / or identification in fungi or yeasts.

[0209] The terms moisturizer, lotion, or body wash refer to low- to medium-viscosity emulsions of oil and water, most commonly oil-in-water, but also water-in-oil, in which the primary benefit in skincare applications is hydrating the skin or reducing its moisture loss. Almost all moisturizers contain a combination of emollients, occlusives, and humectants. Emollients are primarily lipids and oils that hydrate and improve the appearance of the skin. A wide variety of suitable emollients are known and can be used in this article (International Skin Care Ingredient Dictionary and Handbook, edited by Wenninger and McEwen, pp. 1656-61, 1626, and 1654-55 (The Skin care, Toiletry, and Fragrance Assoc., Washington, DC, 7th edition, 1997) (known as the “ICI Handbook” contains examples of many suitable materials). Occlusives (such as petrolatum, lanolin, and beeswax) reduce transepidermal water loss by forming a hydrophobic barrier on the skin. Humectants (such as glycerin and urea) attract water from the external environment and enhance water absorption from the dermis to the epidermis. Additionally, moisturizing formulations may contain emulsifiers to maintain lotion stability and thickeners to achieve the desired viscosity and feel. A variety of other ingredients are often added, such as fragrances, dyes, preservatives, therapeutic agents, proteins, and stabilizers, to obtain other consumer-preferred properties.

[0210] As used herein, the terms “percentage (%) sequence identity” or “percentage (%) sequence similarity” with respect to a reference sequence are defined as the percentage of nucleotide residues in a candidate sequence that are identical to residues in a reference polynucleotide sequence after optimal alignment of the sequences and, where necessary, the introduction of gaps to achieve maximum percentage sequence identity.

[0211] As used herein, the terms “probiotics” or “probiotic microorganisms” are used interchangeably and refer to live microorganisms (including, for example, bacteria or yeast) that, when administered in adequate amounts (topically or orally), beneficially affect the host organism, i.e., by conferring one or more provable benefits upon the host organism. While there is no lower or upper limit to the use of probiotics, it has been shown that at least 10 6 -10 12 Preferably at least 10 6 -10 10 Preferably 10 8 -10 9 CFU as a daily dose will effectively achieve beneficial health effects in the subjects.

[0212] As used in this article, a microbial “strain” refers to a microorganism (such as bacteria or fungi) that remains genetically unchanged during growth or reproduction. This includes many identical microorganisms.

[0213] As used herein, the term "biopure strain" means a strain that does not contain sufficient amounts of other microbial strains to interfere with the replication of the strain or to be detectable by normal techniques. When used in conjunction with the organisms and cultures described herein, "isolation" includes not only biopure strains but also any culture of an organism that grows or is maintained other than those found in nature.

[0214] In one respect, the skin cells described in this article are mammalian skin cells, such as human or animal skin cells.

[0215] As used herein, the terms “sequence identity” or “sequence similarity” mean that two polynucleotide sequences (candidate sequence and reference sequence) are identical (i.e., 100% sequence identity) or similar (i.e., on a nucleotide-by-nucleotide basis) in length of the candidate sequence. When comparing a candidate sequence to a reference sequence, the candidate sequence may contain additions or deletions (i.e., vacancies) compared to a reference sequence used for optimal alignment of the two sequences (which does not contain additions or deletions). Optimal sequence alignment for determining sequence identity can be performed using any number of publicly available local alignment algorithms known in the art (such as ALIGN or Megalign (DNASTAR)) or by inspection.

[0216] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as such lower numerical limit is explicitly stated herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as such higher numerical limit is explicitly stated herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range, as such narrower numerical range is explicitly stated in its entirety herein.

[0217] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0218] Non-limiting examples of the compositions and methods disclosed herein include:

[0219] 1. A skin care composition for providing at least one skin care benefit in a subject in need, said composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein said at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, skin moisturizing (protecting said skin from dehydration by maintaining, restoring and / or improving skin hydration), and any combination thereof.

[0220] 1b. A skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, treating scalp or skin disorders, strengthening the skin barrier, improving skin barrier function, moisturizing the skin (protecting the skin from dehydration by maintaining, restoring and / or improving skin hydration), and any combination thereof.

[0221] 1c. The skin care composition as described in Example 1 or 1b, wherein the skin barrier strengthening comprises applying the composition to the skin or scalp of the subject to strengthen the subject's skin barrier.

[0222] 1d. The skin care composition as described in Example 1 or 1b, wherein the improvement of skin barrier function comprises applying the composition to the skin or scalp of the subject to improve the subject's skin barrier function.

[0223] 1e. The skin care composition as described in Example 1 or 1b, wherein the skin moisturizing comprises applying the composition to the skin or scalp of the subject to moisturize the skin or scalp of the subject.

[0224] 1f. A skin care composition as described in Example 1 or 1b, wherein the composition comprises, in an effective amount relative to the total weight of the composition, about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 3 6%, 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%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% by weight of Bacillus subtilis ferment extract.

[0225] 1g. The skin care composition as described in Example 1, wherein the composition comprises an effective amount relative to the total volume of the composition of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 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%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, up to 100% by volume Bacillus subtilis ferment extract.

[0226] 2. The skin care composition as described in Example 1, wherein the Bacillus subtilis ferment extract is selected from the group consisting of: cell-free supernatant obtained from Bacillus subtilis ferment, cell precipitate extract obtained from Bacillus subtilis ferment, whole broth ferment extract obtained from Bacillus subtilis ferment, and any combination thereof.

[0227] 3. The skin care composition as described in Example 1, wherein the scalp or skin disorder is selected from the group consisting of: scalp microbiota imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, and any combination thereof.

[0228] 4. The skin care composition as described in Example 1, wherein reducing dandruff comprises applying the composition to the skin or scalp of the subject to reduce dandruff.

[0229] 4b. The skin care composition as described in Example 1, wherein reducing the scalp or skin barrier comprises applying the composition to the skin or scalp of the subject to reduce the scalp or skin barrier.

[0230] 5. The skin care composition as described in any of the foregoing embodiments, wherein the Bacillus subtilis ferment extract or fraction thereof is derived from Bacillus subtilis ferment, wherein the Bacillus subtilis is selected from the group consisting of: having a 16S ribosomal RNA sequence (SEQ ID NO: ) similar to that of Bacillus subtilis G01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149857. 1) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. 3) Bacillus subtilis strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, and possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149972. 7) Show at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, and 99.Bacillus subtilis strains exhibiting 16S ribosomal RNA sequence similarity of 5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possess a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0231] 6. The skin care composition as described in any of the foregoing embodiments, wherein the skin care composition further comprises one or more anti-dandruff active agents.

[0232] 7. The skin care composition as described in Example 1, wherein the composition reduces the growth of Malassezia species without affecting the growth of bacteria selected from the group consisting of: Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes strains, and any combination thereof.

[0233] 7b. The skin care composition as described in any of the foregoing embodiments, wherein the composition reduces the growth of Malassezia species without affecting the growth of bacteria selected from the group consisting of: Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes strains, and any combination thereof.

[0234] 8. The skin care composition as described in Example 1, wherein the skin barrier strengthening and / or the improvement of skin barrier function and / or the skin moisturizing occur through fatty acid synthesis, keratinization, synthesis of small proline-rich proteins, or any combination thereof.

[0235] 8a. The skin care composition as described in any of the foregoing embodiments, wherein the skin barrier enhancement occurs through fatty acid synthesis, keratinization, synthesis of small proline-rich proteins, or any combination thereof.

[0236] 8b. The skin care composition as described in Example 8, wherein the fatty acid synthesis occurs via fatty acid elongation.

[0237] 8c. The skin care composition as described in Example 8b, wherein the elongation of the fatty acids occurs by increased expression of the very long chain fatty acid elongase family (ELOVL) fatty acid elongase 7 (ELVOL7).

[0238] 8d. The skin care composition as described in Example 8b, wherein the elongation of the fatty acids occurs by increased expression of the very long chain fatty acid elongase family (ELOVL) fatty acid elongase 7 (ELVOL7), compared to the expression of the gene caused by a control composition identical to the skin care composition except for the absence of the effective amount of the Bacillus subtilis ferment extract or its fraction.

[0239] 8e. The skin care composition as described in Example 8b, wherein the fatty acid synthesis occurs via increased expression of the very long chain fatty acid elongase family (ELOVL) fatty acid elongase 7 (ELVOL7).

[0240] 8f. The skin care composition as described in Example 8b, wherein the fatty acid synthesis occurs by increased expression of the very long chain fatty acid elongase family (ELOVL) fatty acid elongase 7 (ELVOL7), compared to the expression of the gene induced by a control composition identical to the skin care composition except for the absence of the effective amount of the Bacillus subtilis ferment extract or its fraction.

[0241] 8g. The skin care composition as described in Example 8, wherein the keratosis occurs through increased expression of the late keratinization capsule 1c (LCE1C) protein.

[0242] 8h. The skin care composition as described in Example 8, wherein the synthesis of the small proline-rich protein occurs through the increased expression of the small proline-rich protein (SPRR).

[0243] 8i. The skin care composition as described in Example 8, wherein the synthesis of the small proline-rich protein occurs by increased expression of the small proline-rich protein (SPRR), compared to the expression of the gene induced by a control composition identical to the skin care composition except for the absence of the effective amount of the Bacillus subtilis ferment extract or its fraction.

[0244] 8j. The skin care composition as described in Example 8f, wherein the small proline-rich protein is selected from the group consisting of SPRR2F, SPRR2E, SPRR2A, SPRR2D and any combination thereof.

[0245] 9. The skin care composition as described in any of the foregoing embodiments, wherein the skin care composition further comprises additional compounds selected from the group consisting of: fragrances, excipients, preservatives, pH adjusters, aqueous carriers, alcohol carriers, emollients, curing agents, hydrating agents, emulsifiers, solubilizers, surfactants, thickeners, salts, and any combination thereof.

[0246] 10. Use of the skin care composition as described in any of the foregoing embodiments in a cosmetic skin care product.

[0247] 10b. Use of an effective amount of the skin care composition as described in any of the foregoing embodiments in a cosmetic skin care product.

[0248] 11. A skin care product comprising a skin care composition as described in any of the foregoing embodiments and one or more dermatologically or skin care-acceptable components.

[0249] 11b. A skin care product comprising a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0250] 11c. A skin care product comprising a skin care composition for use in reducing skin or scalp disorders, the composition comprising an effective amount of Bacillus subtilis ferment, Bacillus subtilis ferment extract and / or fractions thereof, and one or more dermatologically or skin care-acceptable components, wherein the composition reduces and / or treats the skin or scalp disorder.

[0251] 11d. A skin care product comprising a skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of a fraction of Bacillus subtilis ferment extract or thereof, wherein the Bacillus subtilis is selected from the group consisting of: having a 16S ribosomal RNA sequence (SEQ ID NO: ) similar to that of Bacillus subtilis G01 with accession number CBS149857 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis exhibiting a 16S ribosomal RNA sequence with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, and possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) Bacillus subtilis exhibiting a 16S ribosomal RNA sequence with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity. Bacillus subtilis suitable for this invention also includes: a 16S ribosomal RNA sequence (SEQ ID NO: ) having the same sequence as Bacillus subtilis N01 with accession number CBS149971 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 3) Bacillus subtilis exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence of Bacillus subtilis K01 (SEQ ID NO: 7) deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with number CBS149972 exhibiting at least 97.0%, 97.5%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, and at most 100% sequence similarity.Bacillus subtilis with 16S ribosomal RNA sequences exhibiting 0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possesses a 16S ribosomal RNA sequence (SEQ ID NO: 0.99%) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0252] 12. The skin care product as described in Example 11, wherein the skin care product comprises at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% of the skin care composition by weight relative to the total weight of the skin care product.

[0253] 12b. The skin care product as described in Example 11, wherein the skin care product comprises at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% of the skin care composition by volume relative to the total volume of the skin care product.

[0254] 13. The skin care product as described in Example 11, wherein the product is formulated for topical application.

[0255] 13b. The skin care product as described in Example 11, wherein the product is formulated for topical application to the skin or scalp.

[0256] 14. The skin care product as described in any one of Examples 11-13, wherein the skin care product is selected from the group consisting of: lotions, serums, gels, creams, gels, hydrogels, lotions, solid cosmetics, films, patches, shampoos, loose or dense powders, liquid suspensions or solutions, spray solutions and sticks.

[0257] 14b. A skin care product comprising a first skin care composition and a second skin care composition, wherein the first skin care composition comprises an effective amount of Bacillus subtilis ferment and / or fractions thereof, and wherein the second skin care composition comprises at least an effective amount of at least one second active agent selected from anti-dandruff active agents for topical application.

[0258] 14c. A skin care product comprising a first skin care composition and a second skin care composition, wherein the first skin care composition comprises an effective amount of Bacillus subtilis ferment and / or fractions thereof; wherein the second skin care composition comprises at least an effective amount of at least one second active agent selected from anti-dandruff active agents for topical application, wherein the first skin care composition is formulated into at least one form selected from the group consisting of: gel, lotion, hydrogel, loose or dense powder, liquid suspension or solution, or spray solution.

[0259] 15. A method for providing at least one skin care benefit to a subject in need, the method comprising applying topically to the skin or scalp of the subject a skin care composition or skin care product as described in any of the foregoing embodiments.

[0260] 15b. A method for providing at least one skin care benefit to a subject in need, the method comprising topically applying a skin care composition or skin care product comprising the composition to the skin or scalp of the subject, wherein the composition comprises an effective amount of a fraction of Bacillus subtilis ferment extract or thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0261] 15c. A method for providing at least one skin care benefit to a subject in need, the method comprising topically applying to the subject in need a skin care composition or skin care product as described in any of the foregoing embodiments, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, treating scalp or skin disorders, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0262] 15d. A method for providing at least one skin care benefit to a subject in need, the method comprising topically applying to the subject in need a composition comprising an effective amount of a fraction of Bacillus subtilis ferment extract or thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0263] 16. A method for reducing dandruff in a subject, the method comprising applying topically to the scalp of the subject a skin care composition or skin care product as described in any of the foregoing embodiments.

[0264] 16b. A method for reducing dandruff in a subject, the method comprising topically applying to the subject's scalp a skin care composition or skin care product comprising the skin care composition, wherein the skin care composition comprises an effective amount of Bacillus subtilis ferment extract or a fraction thereof.

[0265] 16c. A method for reducing dandruff in a subject, the method comprising applying topically to the scalp of the subject a skin care composition as described in any of the foregoing embodiments or a skin care product comprising the skin care composition.

[0266] 16d. A method for reducing dandruff in a subject, the method comprising applying topically to the subject's scalp a skin care composition or skin care product containing an effective amount of a fraction of Bacillus subtilis ferment extract or thereof.

[0267] 16e. The method as described in Example 16b, wherein the skin care composition or skin care product is selected from the group consisting of: lotions, serums, gels, creams, emulsions, solid cosmetics, films, patches, and sticks.

[0268] 16f. A method for reducing scalp or skin barriers in a subject, the method comprising topically applying to the subject's scalp or skin a skin care composition or skin care product containing an effective amount of a fraction of Bacillus subtilis ferment extract or thereof.

[0269] 16g. A method for reducing scalp or skin barriers in a subject, the method comprising topically applying a skin care composition or skin care product comprising the skin care composition to the scalp of the subject, wherein the skin care composition comprises an effective amount of Bacillus subtilis ferment extract or a fraction thereof.

[0270] 16h. A method for reducing scalp or skin disorders in a subject, the method comprising topically applying to the subject's scalp a skin care composition or skin care product comprising the skin care composition, wherein the skin care composition comprises an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the scalp or skin disorder is selected from the group consisting of: dandruff (seborrheic dermatitis), scalp flora imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, and any combination thereof.

[0271] 16i. The method as described in Examples 16, 16b, 16c, 16d, 16e, 16f or 16f, wherein the ferment extract reduces the growth of Malassezia species.

[0272] 17. A method for strengthening the skin barrier of a subject, the method comprising applying topically to the skin or scalp of the subject a skin care composition or skin care product as described in any of the foregoing embodiments.

[0273] 17b. A method for strengthening the skin barrier of a subject, the method comprising topically applying to the subject's skin or scalp a skin care composition or skin care product containing an effective amount of Bacillus subtilis ferment extract or a fraction thereof.

[0274] 17c. A method for improving the skin barrier function of a subject, the method comprising applying topically to the skin or scalp of the subject a skin care composition or skin care product as described in any of the foregoing embodiments.

[0275] 17d. A method for improving the skin barrier function of a subject, the method comprising applying topically to the skin or scalp of the subject a skin care composition or skin care product containing an effective amount of Bacillus subtilis ferment extract or a fraction thereof.

[0276] 18. A method for moisturizing the skin of a subject, the method comprising applying topically to the skin or scalp of the subject a skin care composition or skin care product as described in any of the foregoing embodiments.

[0277] 18b. A method for moisturizing the skin of a subject, the method comprising applying topically to the skin or scalp of the subject a skin care composition comprising an effective amount of a fraction of Bacillus subtilis ferment extract or thereof.

[0278] 19. The method of any one of Examples 15-18, wherein the Bacillus subtilis ferment extract is selected from the group consisting of: cell-free supernatant obtained from Bacillus subtilis ferment, cell precipitate extract obtained from Bacillus subtilis ferment, whole broth ferment extract obtained from Bacillus subtilis ferment, and any combination thereof.

[0279] 19b. The method of any one of Examples 15-19, wherein the Bacillus subtilis ferment extract is obtained from a Bacillus subtilis ferment selected from the group consisting of: having a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01 with accession number CBS149857 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. 3) Bacillus subtilis strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, and possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149972. 7) Show at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, and 99.Bacillus subtilis strains exhibiting 16S ribosomal RNA sequence similarity of 6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possess a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0280] 19c. The method, skin care composition, or skin care product as described in any of the foregoing embodiments, wherein the skin care product or skin care composition is selected from the group consisting of: lotions, serums, gels, creams, gels, lotions, solid cosmetics, films, patches, shampoos, and sticks, wherein the skin care product or skin care composition comprises at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% of the skin care composition by weight relative to the total weight of the skin care product.

[0281] 20. A Bacillus subtilis strain variant ferment, Bacillus subtilis strain variant ferment extract, or fraction of said variant ferment, wherein said Bacillus subtilis strain variant is derived from said parent Bacillus subtilis by knocking out at least one sporulation gene of a parent Bacillus subtilis strain, wherein said variant ferment, said variant ferment extract, or said fraction of said variant ferment has increased activity compared to the activity of a ferment, ferment extract, or fraction of said variant ferment, wherein said activity is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0282] 20b. A Bacillus subtilis strain variant ferment, a Bacillus subtilis strain variant ferment extract, or a fraction of said variant ferment, wherein said Bacillus subtilis strain variant is derived from said parent Bacillus subtilis by knocking out the SpoIIE sporulation gene of a parent Bacillus subtilis strain, wherein said variant ferment, said variant ferment extract, or said fraction of said variant ferment has increased activity compared to the activity of a ferment, ferment extract, or fraction of said variant ferment derived from said parent Bacillus subtilis strain, wherein said activity is selected from the group consisting of: reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

[0283] 21. A method for increasing the activity of a Bacillus subtilis strain ferment extract for providing at least one skin care benefit, the method comprising: (a) providing a parent Bacillus subtilis strain capable of producing an effective amount of the ferment extract for providing at least one skin care benefit selected from the group consisting of: reduced dandruff, reduced scalp or skin barrier, strengthened skin barrier, improved skin barrier function, skin hydration, and any combination thereof; (b) modifying the parent Bacillus subtilis strain by knocking out at least one spore-forming gene from the genome of the Bacillus subtilis strain, thereby producing a variant strain that cannot form spores; and (c) producing a ferment extract of the Bacillus subtilis variant strain, wherein the ferment extract of the Bacillus subtilis variant strain has increased activity compared to the activity of the ferment extract of the parent Bacillus subtilis strain, for providing at least one skin care benefit selected from the group consisting of: reduced dandruff, reduced scalp or skin barrier, strengthened skin barrier, improved skin barrier function, skin hydration, and any combination thereof in a subject in need.

[0284] 21b. A method for increasing the activity of a Bacillus subtilis strain ferment extract for providing at least one skin care benefit, the method comprising: (a) providing a parent Bacillus subtilis strain capable of producing an effective amount of the ferment extract for providing at least one skin care benefit selected from the group consisting of: reduced dandruff, reduced scalp or skin barrier, strengthened skin barrier, improved skin barrier function, skin hydration, and any combination thereof; (b) modifying the parent Bacillus subtilis strain by knocking out the SpoIIE sporulation gene from the genome of the Bacillus subtilis strain, thereby producing a variant strain that cannot form sporulations; and (c) The fermentation extract of the Bacillus subtilis variant strain, wherein the fermentation extract of the Bacillus subtilis variant strain has increased activity when compared with the activity of the fermentation extract of the parent Bacillus subtilis strain, is intended to provide at least one skin care benefit selected from the group consisting of: reduced dandruff, reduced scalp or skin barrier, strengthened skin barrier, improved skin barrier function, skin hydration, and any combination thereof in subjects in need.

[0285] 22. A skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa.

[0286] 23. A skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa by weight, from 0.1% to 100% relative to the total weight of the effective amount.

[0287] 24. A skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa by volume, from 0.1% to 100% of the total volume relative to the effective amount.

[0288] 25. The skin care composition as described in Examples 22-24, wherein the at least one active agent is bacitracin.

[0289] 26. A skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reduction of dandruff, reduction of scalp or skin barriers, Malassezia growth inhibition, reduction of Malassezia species growth without affecting the growth of Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes strains, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa.

[0290] 27. A skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reduction of dandruff, reduction of scalp or skin barriers, Malassezia growth inhibition, reduction of Malassezia species without affecting the growth of Staphylococcus aureus and Propionibacterium acnes strains, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa by volume, from 0.1% to 100% of the total volume relative to the effective amount.

[0291] 28. A skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reduction of dandruff, reduction of scalp or skin barriers, Malassezia growth inhibition, reduction of Malassezia species without affecting the growth of Staphylococcus aureus and Propionibacterium acnes strains, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent of less than 3 kDa in weight, from 0.1% to 100% relative to the total weight of the effective amount.

[0292] 29. The skin care composition as described in Examples 26-28, wherein the at least one active agent is bacitracin.

[0293] 30. A skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: skin barrier strengthening, improved skin barrier function, skin moisturizing, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises at least one active agent selected from the group consisting of: less than 100 kDa, less than 50 kDa, less than 30 kDa, less than 10 kDa, less than 3 kDa, and any combination thereof.

[0294] 31. A skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, and wherein the effective amount of the Bacillus subtilis ferment extract comprises an active agent of less than 3 kDa.

[0295] 32. The skin care composition of any one of Examples 22-31, wherein the ferment extract or fraction thereof comprises a ferment extract or fraction thereof from the microorganism Bacillus subtilis, wherein the Bacillus subtilis is selected from the group having a 16S ribosomal RNA sequence (SEQ ID NO: ) similar to that of Bacillus subtilis G01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149857. 1) A *Bacillus subtilis* strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of *Bacillus subtilis* PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149969. 2) Bacterial strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. 3) Bacterial strains exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149972. 7) Show at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, and 99.Bacterial strains with 16S ribosomal RNA sequences exhibiting 5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possessing a 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity. Example

[0296] In the following examples, unless otherwise stated, parts and percentages are by weight, and degrees are in degrees Celsius. It should be understood that while these examples illustrate embodiments of this disclosure, they are given by way of illustration only. From the foregoing discussion and these examples, those skilled in the art can make various changes and modifications to this disclosure to adapt it to various uses and conditions. Such modifications are also intended to fall within the scope of the appended claims.

[0297] The following abbreviations in this specification correspond to units of measurement, techniques, properties, or compounds, as follows: “sec” or “s” means second, “min” means minute, “h” or “hr” means hour, “μL” means microliter, “mL” means milliliter, “L” means liter, “mM” means millimole, “M” means mole, “mmol” means millimole, “ppm” means parts per million, “wt” means weight, “wt%” means weight percentage, “g” means gram, “mg” means milligram, “μg” means microgram, “ng” means nanogram, “concentration” means concentration, and “Trt” means treatment.

[0298] Example 1

[0299] Bacillus subtilis strain

[0300] To obtain various Bacillus strains, various fermented foods and beverages were treated by heating at 80°C for 20 minutes. After heat treatment, aliquots were spread onto tryptic soy broth agar plates. Each liter of tryptic soy broth contained 17 g of tryptic peptone, 3 g of soy peptone, 5 g of sodium chloride, 2.5 g of dipotassium hydrogen phosphate, and 2.5 g of dextrose. Phylogenetic identity of each Bacillus strain was determined by sequencing the 16S region using primer set 8F (AGA GTT TGA TYM TGG CTC; SEQ ID NO: 4) and 515F (GTGCCA GCM GCC GCG GTA AA; SEQ ID NO: 5), where "M" stands for adenine or cytosine nucleotides. The 16S region was amplified using primer set 8F and 1492R (CGG TTA CCTTGT TAC GAC TT; SEQ ID NO: 6).

[0301] The Bacillus species applicable to this invention include, but are not limited to, Bacillus species selected from the group consisting of: those having a 16S ribosomal RNA sequence (SEQ ID NO: 1) of Bacillus subtilis G01 with accession number CBS149857 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to the 16S ribosomal RNA of Bacillus subtilis G01 with accession number CBS149857 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). The Bacillus subtilis strain with a 16S ribosomal RNA sequence has a 98.75% nucleotide identity across its entire genome with Bacillus subsp. *bacillus* PC01 (which shares 98.75% nucleotide identity with the subspecies *Bacillus subtilis* quosorum, and can therefore also be classified as a *Bacillus subtilis* quosorum type strain) deposited at the Westdick Institute for Fungal Biodiversity (WFDB). ID NO: 2) A Bacillus subtilis strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity to a 16S ribosomal RNA sequence, possessing a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis strain N01 with accession number CBS149971 deposited at the Westdick Institute for Fungal Biodiversity (WFDB). NO:3) A Bacillus subtilis strain exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% 97.0% sequence similarity to a 16S ribosomal RNA sequence of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) (SEQ ID NO: 7) exhibiting at least 97.0%.Bacillus subtilis strains exhibiting 16S ribosomal RNA sequence similarity of 0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, possess a 16S ribosomal RNA sequence (SEQ ID NO: 0.99%) similar to that of Bacillus subtilis G01_spo, deposited at the Westdick Institute for Fungal Biodiversity (WFDB). 1) Bacillus subtilis strains, or any combination thereof, exhibiting 16S ribosomal RNA sequences with at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and at most 100% sequence similarity.

[0302] Additionally, Bacillus subtilis 168 (ATCC23857) with the 16S ribosomal RNA sequence of SEQ ID NO: 8 was used for comparison.

[0303] Example 2

[0304] Bacillus subtilis ferment

[0305] Bacillus subtilis strains were grown in SUM (soybean peptone-urea micronutrient) medium. Each liter of SUM medium (1.0X strength) consisted of 10 g soybean peptone, 75 g glucose, 3 ml dipotassium hydrogen phosphate (1 M), 3.6 g urea, and 10 ml of micronutrient stock solution. Each liter of micronutrient stock solution contained 1.47 g sodium citrate·2H₂O, 1.47 g CaCl₂·2H₂O, 0.4 g FeSO₄·7H₂O, 0.1 g MnSO₄·H₂O, 0.1 g ZnSO₄·H₂O, 0.05 g CuCl₂·2H₂O, 0.1 g CoCl₂·6H₂O, and 0.1 g Na₂MoO₄·2H₂O. The SUM medium was adjusted to pH 7.3. Bacillus subtilis fermentation was carried out in 125 ml flat-bottomed shake flasks containing 25 ml of the medium. The flask was placed in a platform shaker at a constant speed of 200 rpm at 32°C. Overnight fresh seed culture was used as inoculum, with an initial OD of 0.125 at 600 nm. The culture was allowed to grow for 48 hours.

[0306] Bacillus subtilis can also be grown in TSB (trypsinized soybean broth) medium (TSG) supplemented with 1% glucose. Each liter of TSG medium (1.0X strength) consists of 17 g tryptone, 3 g soybean peptone, 5 g sodium chloride, 10 g glucose, and 2.5 g dipotassium hydrogen phosphate. The TSG medium can be adjusted to pH 7.3. Although SUM medium is used as the growth medium for Bacillus subtilis as described herein, Bacillus subtilis strains can be grown in any suitable medium that can support growth. Furthermore, incubation time can vary depending on the growth medium or growth method. It should be understood that fermentation products can be collected at different incubation hours and still produce fermentation products containing a viable amount of Bacillus subtilis fermentation product extract.

[0307] Example 3

[0308] Bacillus subtilis ferment extract

[0309] The following describes how to obtain an extract from Bacillus subtilis fermentation.

[0310] Cell-free supernatant of Bacillus subtilis fermentation

[0311] Cells were removed from the Bacillus subtilis ferment by centrifuging at 4,000 g for 20 min to precipitate the cells. The resulting cell-free supernatant (fermentation extract) was sterilized by passing it through a 0.2 µM filter.

[0312] Cell precipitate extract of Bacillus subtilis fermentation

[0313] Cell precipitate extract from Bacillus subtilis fermentation was obtained by precipitating cells (centrifuging at 4,000 to 8,000 xg) and discarding the supernatant to leave a partial cell precipitate. The precipitate was resuspended in acidic water (1 / 10 v / v) to pH 2.5–4.0, vortexed, precipitated by centrifugation (4,000 to 8,000 xg), and the extract was passed through a 0.2 µM filter.

[0314] Starting with cell precipitation has the following advantages: the volume of the resuspended liquid (extract volume) can be determined to produce an effective amount of active extract and / or further concentrate it to produce an effective amount of active extract. Furthermore, resuspending the precipitate in water or a non-fermented broth liquid results in a reduction or elimination of inactive components in the fermented broth that may interfere with the activity of the cell-precipitated extract.

[0315] Combination of cell-free supernatant and cell precipitate extract of Bacillus subtilis fermentation

[0316] Bacillus subtilis fermentation extract can also be produced by combining the above-mentioned cell-free supernatant fraction with the above-mentioned cell precipitate extract.

[0317] Whole meat broth Bacillus subtilis ferment extract

[0318] To obtain the whole broth fermentation extract, the Bacillus subtilis fermentation extract was prepared as follows: First, the pH of the fermentation product (total fermentation broth - Example 2) was adjusted to pH 2.5-4.0, then insoluble cell material was precipitated, and the supernatant was filtered through a 0.2 µM filter to obtain a pH-adjusted cell-free supernatant. Alternatively, the Bacillus subtilis whole broth fermentation extract was prepared as follows: First, the pH of the fermentation product (total fermentation broth) was adjusted to an alkaline pH, then insoluble cell material was precipitated, and optionally the supernatant was filtered through a 0.2 µM filter to obtain a cell-free supernatant (also known as the "whole broth fermentation extract").

[0319] Example 4

[0320] Bacillus subtilis ferment extract inhibits the growth of Malassezia species.

[0321] The ability of Bacillus subtilis ferment extract to inhibit the growth of Malassezia species was evaluated. Bacillus subtilis G01 (CBS149857), Bacillus subtilis N01 (CBS149971), Bacillus subtilis K01 (CBS149972), and Bacillus subtilis PC01 (CBS149969) were grown in SUM (soybean peptone-urea micronutrient) medium. Each liter of SUM medium (1.0 X strength) consisted of 10 g soybean peptone, 75 g glucose, 3 ml dipotassium hydrogen phosphate (1 M), 3.6 g urea, and 10 ml of the micronutrient stock solution. Each liter of the micronutrient stock solution contained 1.47 g sodium citrate·2H₂O, 1.47 g CaCl₂·2H₂O, 0.4 g FeSO₄·7H₂O, 0.1 g MnSO₄·H₂O, 0.1 g ZnSO₄·H₂O, 0.05 g CuCl₂·2H₂O, 0.1 g CoCl₂·6H₂O, and 0.1 g Na₂MoO₄·2H₂O. The SUM medium was adjusted to pH 7.3. A 0.5x strength SUM medium was obtained by diluting the medium by half with purified water. The Bacillus subtilis ferment was carried out in 125 ml flat-bottomed shake flasks containing 25 ml of 0.5x strength SUM medium. The flasks were placed in a platform shaker at a constant speed of 200 rpm at 32°C. Overnight fresh seed culture was used as inoculum, with an initial OD of 0.125 at 600 nm. After 48 hours of growth, the fermentation broth was sterilized by centrifugation and filtration to obtain a cell-free supernatant (fermentation broth extract). For comparative purposes, Bacillus subtilis 168 (ATCC23857) was used and grown under the same conditions.

[0322] To determine the growth-inhibiting activity of Bacillus subtilis ferment extract, *Malassezia furfur* (M. furfur) was used as the target organism in an inhibition zone assay using agar plates. *Malassezia furfur* ATCC 14521 was cultured prior to the growth-inhibiting activity test as follows: It was inoculated 1:100 from frozen stock solution into mDixon medium containing 500 µg / mL chloramphenicol and incubated for 2 days at 32°C in a steady-state shaker at 250 rpm. Each liter of mDixon medium contained 36 g malt extract, 20 g dried ox bile, 10 ml Tween-40, 6 g peptone, 2 ml glycerol, and 2 ml oleic acid. The pH of the medium was adjusted to 6. Fresh *Malassezia furfur* cells were suspended in mDixon broth to obtain an optical density equivalent to 0.4 OD. Agar plates were uniformly inoculated with 100 µl aliquots. After drying, 5 µl of the Bacillus subtilis fermentation extract was directly spotted onto the top of an agar plate. The plate was incubated at 32°C for 48 hours. The presence of a clear zone indicated growth inhibition, and the size of the inhibition zone was used as an indicator of the efficacy of the fermentation extract. The size of the zone was measured based on its diameter in millimeters (mm). The results are shown in Table 1.

[0323] Table 1. Malassezia strains obtained from fermentation extracts of different Bacillus strains using inhibition zone assays. Growth inhibition assay.

[0324]

[0325] As shown in Table 1, the fermentation extract obtained from Bacillus subtilis strain 168 (ATCC 23857) showed no growth inhibition against Malassezia, while the fermentation extracts from Bacillus subtilis PC01 and Bacillus subtilis N01 exhibited good growth inhibitory activity. The fermentation extracts from Bacillus subtilis strains G01 and K01 had larger inhibition zones, also indicating high efficacy in inhibiting Malassezia growth.

[0326] Human skin is colonized by a diverse microbiome (Byrd, A., Belkaid, Y. & Segre, J. The human skin microbiome. Nat Rev Microbiol 16, 143-155; 2018). Staphylococcus aureus and *Dermatobacterium* (formerly known as *Propionibacterium*) are two dominant commensal species. To evaluate the specificity of the ferment extract of *Bacillus subtilis* G01 against other skin microorganisms, *Staphylococcus aureus* ATCC 27844, *Staphylococcus epidermidis* ATCC 12228, and *Dermatobacter acnes* K2 strain were used as target organisms in an inhibition zone assay. Direct spotting of 5 µl of the ferment extract of *Bacillus subtilis* G01 did not result in a visible clear zone against either organism. The results indicate that the fermentation extract produced by Bacillus subtilis G01 has high specificity for the yeast Malassezia spp., without affecting the growth of Staphylococcus aureus, Staphylococcus epidermidis, and Propionibacterium acnes strains.

[0327] Example 5

[0328] Use Bacillus subtilis ferment extract to improve skin barrier function.

[0329] The ability of Bacillus subtilis ferment extract to improve skin barrier function was evaluated. Bacillus subtilis strain G01 (CBS149857) was grown in TSB (trypsinized soybean broth) medium (TSG) supplemented with 1% glucose. Each liter of TSG medium (1.0 X strength) consisted of 17 g tryptone, 3 g soybean peptone, 5 g sodium chloride, 10 g glucose, and 2.5 g dipotassium hydrogen phosphate. The TSG medium was adjusted to pH 7.3. The fermentation of Bacillus subtilis was carried out in 125 ml flat-bottomed shake flasks containing 25 ml of medium. The flasks were placed in a platform shaker at a constant speed of 200 rpm at 32°C. Overnight fresh seed culture was used as inoculum, with an initial OD of 0.125 at 600 nm. After 24 hours of growth, cell-free supernatant (ferment extract) was obtained by centrifugation and sterilization by filtration.

[0330] Normal human epidermal keratinocytes (NHEK) isolated from a single adult donor were thawed in keratinocyte growth medium 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), adrenaline (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). The NHEK cells were then cultured in T75 flasks containing KGM2 to achieve 75%–80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, the NHEK cells were removed from the T75 flasks using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK cells were resuspended in KGM2, counted using a hemocytometer, and seeded into transwell chambers (6.5 mm chambers; 0.4 μm polyester membrane; tissue culture-treated polystyrene 24-well plates). NHEK cells were incubated in a 37°C incubator with 5% CO2 and 95% humidity until 100% confluence on the transwell membrane. At this point, the medium was aspirated, and NHEK cells were subsequently grown in KGM2 supplemented with 1.5 mM (instead of 0.06 mM) calcium chloride for the remainder of the experiment. Bacillus subtilis G01 fermentation extract was added to the top wells to bring the final fermentation extract concentration to 0.1%. Transepithelial resistance (TEER) was measured daily using an epithelial voltammometer / ohmmeter (EVOM) after the addition of the fermentation extract. The medium and fermentation extract were refreshed every 2 days. The results are shown in Table 2.

[0331] Table 2. Bacillus subtilis G01 fermentation extract increases transepithelial resistance (TEER).

[0332]

[0333] The results in Table 2 show that, compared with the untreated (without fermentation extract) control, the addition of 0.1% Bacillus subtilis G01 fermentation extract resulted in a higher percentage increase in TEER value, indicating that skin barrier function was improved and the skin barrier was strengthened.

[0334] Example 6

[0335] The fraction of Bacillus subtilis ferment extract is used to improve skin barrier function.

[0336] The ability of specific molecular weight fractions of Bacillus subtilis ferment extract to improve skin barrier function was evaluated. Ferment extracts from Bacillus subtilis strain G01 (CBS149857) were produced using the same methods described in Example 5. After centrifugation and filtration sterilization, the cell-free supernatant (ferment extract) was molecularly fractionated by passing the supernatant through a series of molecular weight cutoff filters in the following order: 100 kilodaltons (kDa), 50 kDa, 30 kDa, and 10 kDa. Ferment extracts that did not pass through the filters were retained and referred to as the residue (R), and ferment extracts that passed through the filters were retained and referred to as the effluent (FT). As performed in Example 5, the ability of each Bacillus subtilis G01 molecular weight residue and effluent to improve skin barrier function compared to an untreated (no ferment extract) control was tested using NHEK. The results are shown in Table 3A.

[0337] Table 3A. Fermentation extract fractions of various Bacillus subtilis G01 molecular weights show an increase in transepithelial resistance (TEER).

[0338]

[0339] The results in Table 3A show that, in the TEER assay, the addition of Bacillus subtilis G01 ferment extract (cell-free supernatant) at a concentration of 0.1% through 100 kDa, 50 kDa, and 30 kDa molecular weight cutoff filters resulted in a higher percentage increase in TEER values ​​compared to the untreated (no ferment extract) control, indicating improved skin barrier function and a strengthened skin barrier.

[0340] We further evaluated the ability of the 10 kDa effluent to improve skin barrier function by first concentrating the 10 kDa effluent tenfold (10X) and then adding it to the TEER assay. Briefly, aliquots of the 10 kDa effluent ferment extract were centrifuged under vacuum until the final volume reached 10% of the initial volume. This fraction was designated 10 kDa effluent 10X. The ability of the 10 kDa effluent 10X samples to improve skin barrier function compared to the untreated (no ferment extract) control was then tested in a dose-dependent manner (resulting in final assay concentrations of 0.05%, 0.1%, 0.2%, and 0.4% of the initial concentrated ferment extract). Taking into account the concentration factor, the final assay concentrations of 0.05%, 0.1%, 0.2%, and 0.4% represent 0.5%, 1%, 2%, and 4% of the unconcentrated ferment extract. The results are shown in Table 3B.

[0341] Table 3B. < 10 kDa effluent 10X Bacillus subtilis fermentation extract fraction increased transepithelial Resistor (TEER).

[0342]

[0343] The results in Table 3B indicate that the concentrated form of Bacillus subtilis G01 ferment extract (less than 10 kDa) can increase the TEER value compared to the untreated (no ferment extract) control, suggesting improved skin barrier function.

[0344] Example 7.

[0345] Improve skin barrier function through heat-sensitive Bacillus subtilis ferment extract.

[0346] The ability of heat-sensitive Bacillus subtilis ferment extract to improve skin barrier function was evaluated. Ferment extracts (cell-free supernatant) from Bacillus subtilis strain G01 (CBS149857) were produced using the same method as described in Example 5. The Bacillus subtilis G01 ferment extract was incubated at 100°C for 10 minutes prior to use in the assay. Subsequently, the ability of untreated and heat-treated Bacillus subtilis G01 ferment extracts to improve skin barrier function was tested using NHEK as described in Example 5 (Table 4). The results were compared with an untreated (no ferment extract) control.

[0347] Table 4. The Bacillus subtilis G01 component that enhances the keratinocyte barrier is heat-sensitive.

[0348]

[0349] The results in Table 4 demonstrate that heating at 100°C did not increase the keratinocyte barrier of Bacillus subtilis G01 ferment extract.

[0350] Example 8.

[0351] Improve skin barrier function using fermentation extracts from multiple Bacillus subtilis strains

[0352] The ability of fermentation extracts from various Bacillus subtilis strains to improve skin barrier function was evaluated. Fermentation extracts (cell-free supernatant) from Bacillus subtilis strains G01 (CBS149857), Bacillus subtilis 168 (ATCC23857), Bacillus subtilis N01 (CBS149971), and Bacillus subtilis K01 (CBS149972) were produced using the same methods as described in Example 5. The ability of each Bacillus subtilis fermentation extract to improve skin barrier function compared to an untreated (no fermentation extract) control was tested using NHEK, as performed in Example 5.

[0353] Table 5. Fermentation extracts (cell-free supernatant) of Bacillus subtilis strains G01 and N01 Increase transepithelial resistance (TEER).

[0354]

[0355] The results shown in Table 5 demonstrate that the ferment extracts (cell-free supernatant) of Bacillus subtilis G01 and Bacillus subtilis N01 increased transepithelial resistance (TEER), indicating that skin barrier function was improved and the skin barrier was strengthened.

[0356] Example 9.

[0357] Bacillus subtilis ferment extract strengthens the skin barrier through fatty acid synthesis.

[0358] The mechanism by which Bacillus subtilis ferment extract affects the keratinocyte barrier was investigated. Normal human epidermal keratinocytes (NHEK) isolated from a single adult donor were thawed in keratinocyte growth medium 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), adrenaline (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). The NHEK cells were then cultured in T75 flasks containing KGM2 to achieve 75%–80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, the NHEK cells were removed from the T75 flasks using trypsin and centrifuged at 1500 RPM for 5 min at room temperature (RT). NHEK cells were resuspended in KGM2, counted using a hemocytometer, and seeded into 96-well plates. A fermentation extract (cell-free supernatant) from Bacillus subtilis G01 (CBS149857) was produced as described in Example 5. Cells were incubated overnight at 37°C with 5% CO2 and 95% humidity, and then stimulated with the Bacillus subtilis G01 fermentation extract for 18 hours. Following stimulation with the Bacillus subtilis G01 fermentation extract, RNA was isolated from NHEK cells using the RNAqueous Micro Kit. The RNA was further processed using the Illumina Chain-Specific mRNA Preparation Kit to extract mRNA from all other RNA types. Individual mRNA libraries were normalized to 2 nM, merged, and 4% phiX was added to the merged mRNA libraries. A (1) nM merged library was loaded onto an Inmena chip (P1, P2, or P3) and then sequenced using primer A (SEQ ID NO: 9) and primer B (SEQ ID NO: 10) with an Inmena NextSeq 1000 or Inmena NextSeq 2000 instrument. The results are shown in Table 6.

[0359] Table 6. Fermentation extracts of Bacillus subtilis G01 showed increased levels of the NHEK long-chain fatty acid elongase family. Expression of (ELOVL) fatty acid elongase 7 (ELOVL7).

[0360]

[0361] The results presented in Table 6 demonstrate that the fermentation extract (cell-free supernatant) of Bacillus subtilis G01 increased the expression of the ELVOL7 gene (gene ID 79993) in NHEK, indicating that Bacillus subtilis G01 strengthens the keratinocyte barrier (skin barrier) by extending fatty acids, and is therefore suitable for strengthening the skin barrier, moisturizing the skin, reducing dandruff, and reducing skin and scalp barriers.

[0362] Example 10.

[0363] Bacillus subtilis ferment extract strengthens the skin barrier through keratinization.

[0364] The mechanism by which Bacillus subtilis ferment extract affects the keratinocyte barrier was analyzed. Normal human epidermal keratinocytes (NHEK) isolated from a single adult donor were thawed in keratinocyte growth medium 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), adrenaline (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). The NHEK cells were then cultured in T75 flasks containing KGM2 to achieve 75%–80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, the NHEK cells were removed from the T75 flasks using trypsin and centrifuged at 1500 RPM for 5 min at room temperature (RT). NHEK cells were resuspended in KGM2, counted using a hemocytometer, and seeded into 96-well plates. A fermentation extract (cell-free supernatant) from Bacillus subtilis G01 (CBS149857) was produced as described in Example 5. Cells were incubated overnight at 37°C with 5% CO2 and 95% humidity, and then stimulated with the Bacillus subtilis G01 fermentation extract for 18 hours. Following stimulation with the Bacillus subtilis G01 fermentation extract, RNA was isolated from NHEK cells using the RNAqueous Micro Kit. The RNA was further processed using the Inmena Chain-Specific mRNA Preparation Kit to extract mRNA from all other RNA types. Individual mRNA libraries were normalized to 2 nM, merged, and 4% phiX was added to the merged mRNA libraries. The 1 nM merged library was loaded onto the P1, P2, or P3 Imina chip and then sequenced using primer A (SEQ ID NO: 9) and primer B (SEQ ID NO: 10) using an Imina NextSeq 1000 or Imina NextSeq 2000 instrument. The results are shown in Table 7.

[0365] Table 7. Fermentation extract of Bacillus subtilis G01 (cell-free supernatant) increased late keratinization in NHEK. Expression of membrane 1c (LCE1C)

[0366]

[0367] The results presented in Table 7 demonstrate that the fermentation extract (cell-free supernatant) of Bacillus subtilis G01 increased the expression of the LCE1C gene (gene ID 353133) in NHEK, indicating that Bacillus subtilis G01 strengthens the keratinocyte barrier (skin barrier) through increased keratinization, and is therefore suitable for strengthening the skin barrier, moisturizing the skin, reducing dandruff, and reducing skin and scalp barriers.

[0368] Example 11.

[0369] Bacillus subtilis G01 ferment extract improves the skin barrier by synthesizing small proline-rich proteins.

[0370] The mechanism by which Bacillus subtilis ferment extract affects the keratinocyte barrier was analyzed. Normal human epidermal keratinocytes (NHEK) isolated from a single adult donor were thawed in keratinocyte growth medium 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), adrenaline (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). The NHEK cells were then cultured in T75 flasks containing KGM2 to achieve 75%–80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, the NHEK cells were removed from the T75 flasks using trypsin and centrifuged at 1500 RPM for 5 min at room temperature (RT). NHEK cells were resuspended in KGM2, counted using a hemocytometer, and seeded into 96-well plates. A fermentation extract (cell-free supernatant) from Bacillus subtilis G01 (CBS149857) was produced as described in Example 5. Cells were incubated overnight at 37°C with 5% CO2 and 95% humidity, and then stimulated with the Bacillus subtilis G01 fermentation extract for 18 hours. Following stimulation with the Bacillus subtilis G01 fermentation extract, RNA was isolated from NHEK cells using the RNAqueous Micro Kit. The RNA was further processed using the Inmena Chain-Specific mRNA Preparation Kit to extract mRNA from all other RNA types. Individual mRNA libraries were normalized to 2 nM, merged, and 4% phiX was added to the merged mRNA libraries. The 1 nM merged library was loaded onto the P1, P2, or P3 Imina chip and then sequenced using primer A (SEQ ID NO: 9) and primer B (SEQ ID NO: 10) using an Imina NextSeq 1000 or Imina NextSeq 2000 instrument. The results are shown in Table 8.

[0371] Table 8. Fermentation extract of Bacillus subtilis G01 (cell-free supernatant) showed increased levels of small proline in NHEK. Expression of SPRR2F, SPRR2E, SPRR2A and SPRR2D proteins.

[0372]

[0373] The results presented in Table 8 demonstrate that Bacillus subtilis G01 increases the expression of the genes SPRR2F (gene ID 6705), SPRR2E (gene ID 6704), SPRR2A (gene ID 6700), and SPRR2D (gene ID 6703) in NHEK, indicating that Bacillus subtilis G01 strengthens (increases) the keratinocyte barrier through the synthesis of small proline-rich proteins, and is therefore suitable for reducing scalp disorders such as dandruff and seborrheic dermatitis, as well as for strengthening the skin barrier and moisturizing the skin.

[0374] Example 12

[0375] Knocking out the spoIIE gene from Bacillus subtilis further enhances the extract of Bacillus subtilis ferment. The ability to improve skin barrier function.

[0376] Bacillus subtilis strains contain genes associated with spore formation, such as SpollE (SEQ ID NO: 11; Barak et al. 1996, Molecular Microbiology 19(5), 1047-1060). On the one hand, there is a desire to eliminate the SpollE gene, especially for Bacillus subtilis producing strains.

[0377] To evaluate the ability of Bacillus subtilis G01 spore-forming spoIIE knockout (referred to as Bacillus subtilis G01_spo, CBS151098) ferment extract to improve skin barrier function.

[0378] A CRISPR-Cas9 plasmid (designated pMY1-3) was constructed to inhibit the spoIIE locus from Bacillus subtilis G01 (CBS147469) by deleting its spore-forming ability. The pMY1-3 plasmid contains a Streptococcus pyogenes Cas9 endonuclease expression cassette, an editable template insert region (SEQ ID NO: 12), and a kanamycin resistance cassette containing a Bacillus subtilis G01 homolog and sgRNA (SEQ ID NO: 13). The pMY1-3 plasmid was electroporated into Endura™ electrocompetent cells (Biosearch Technologies). The assembled construct was validated via Sanger sequencing. pMY1-3 was amplified using rolling circle amplification (RCA) with the GenomiPhi HY Ready-To-Go DNA Amplification Kit (Cytiva).

[0379] Bacillus subtilis G01 (CBS147469) was modified to contain the competent plasmid pBL.comK as described in WO 2019 / 40423 and to lack the spoIIE gene. Briefly, pBL.comK was transformed into Bacillus subtilis G01 using naturally competent cells and spectinomycin as a resistance marker. The pBL.comK-containing strain was then made competent as described in WO 2021 / 146411 and subsequently transformed with plasmid pMY1-3. 200 µL of competent cells was mixed with 50 µL of pMY1-3RCA product and 1 mL of Luria-Bertani (LB) broth and incubated at 30°C and 250 RPM for 1.5 h. The mixture was plated onto LB agar plates containing 20 ppm kanamycin to select cells transformed with the Cas9 plasmid pMY1-3. Isolates were screened for spoIIE deletions using Q5® PCR primers BSspo-check-del-F (SEQ ID NO: 14) and BSspo-check-del-R (SEQ ID NO: 15). Colonies with the spoIIE deletion (ΔspoIIE) produced a 2448 bp PCR product, while wild-type strains containing the complete spoIIE gene produced a 4932 bp PCR product. The deletion was confirmed by Sanger sequencing, and the confirmed mutant isolates were stored in glycerol stock solution at -80°C.

[0380] The pBL.comK and pMY1-3 components in isolates were eliminated by culturing them in antibiotic-free TSA for three days, subculturing them every 24 hours, and then plated onto plain trypsinized soybean agar (TSA). Colonies from the TSA were selected and inoculated onto antibiotic-free LB agar, LB agar containing 20 ppm kanamycin, and LB agar containing 100 ppm spectinomycin. The eliminated isolates grew on plain LB agar and failed to grow on LB agar containing kanamycin or spectinomycin.

[0381] After confirming the spoIIE deletion genotype, phenotypic analysis was performed using heat treatment to confirm the loss of sporulation ability. *Bacillus subtilis* G01 was grown in Difco sporulation medium (DSM) to promote sporulation (Nicholson and Setlow, 1990). Wild-type and ΔspoIIE clones were freshly plated on TSA, and single colonies were inoculated into DSM and incubated for three days at 32°C and 200 RPM. Two 100 µL aliquots were taken from each culture and transferred to 1.5 mL microcentrifuge tubes. The test aliquots were heated at 90°C for 15 minutes, while the control aliquots were kept at room temperature (RT). All 100 µL aliquots were plated onto TSA and incubated overnight at 32°C. Sporulation-positive isolates (such as wild-type *Bacillus subtilis* G01) showed growth at both room temperature (RT) and 90°C treatments. Spore-forming negative isolates showed growth at RT but not at 90°C. Spore-forming negative clones (also known as Bacillus subtilis G01_spo strain) were stored in glycerol stock solution at -80°C.

[0382] Bacillus subtilis G01 (CBS147469, also known as Bacillus subtilis G01 wild-type (WT)) and Bacillus subtilis G01_spo strain were grown in TSB (trypsinized soybean broth) medium (TSG) supplemented with 1% glucose. Each liter of TSG medium (1.0 X strength) consisted of 17 g tryptone, 3 g soybean peptone, 5 g sodium chloride, 10 g glucose, and 2.5 g dipotassium hydrogen phosphate. The TSG medium was adjusted to pH 7.3. The production of Bacillus subtilis G01 WT and KO ferments was carried out in 125 ml flat-bottomed shake flasks containing 25 ml TSG. The flasks were placed in a platform shaker at a constant speed of 200 rpm at 32°C. Overnight fresh seed culture was used as inoculum, with an initial OD of 0.125 at 600 nm. After 24 hours of growth, the ferment broth was sterilized by centrifugation and filtration to obtain the ferment broth extract (cell-free supernatant).

[0383] Normal human epidermal keratinocytes (NHEK) isolated from a single adult donor were thawed in keratinocyte growth medium 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), adrenaline (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). The NHEK cells were then cultured in T75 flasks containing KGM2 to achieve 75%–80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, the NHEK cells were removed from the T75 flasks using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK cells were resuspended in KGM2, counted using a hemocytometer, and seeded into transwell chambers (6.5 mm chambers; 0.4 μm polyester membrane; tissue culture-treated polystyrene 24-well plates). NHEK cells were incubated in a 37°C incubator with 5% CO2 and 95% humidity until 100% confluence on the transwell membrane. At this point, the medium was aspirated, and NHEK cells were subsequently grown in KGM2 supplemented with 1.5 mM (instead of 0.06 mM) calcium chloride for the remainder of the experiment. Bacillus subtilis ferment broth extract was added to the top wells to bring the final extract concentration to 0.1% by volume. Transepithelial resistance (TEER) was measured daily using an epithelial voltammometer / ohmmeter (EVOM) after the addition of the ferment broth extract. The medium and ferment broth extract were refreshed every 2 days. The results are shown in Table 9 and compared with an untreated control (without ferment broth extract).

[0384] Table 9. Spore formation of Bacillus subtilis ferment extract (cell-free supernatant) by spoIIE knockout. Increase transepithelial resistance (TEER)

[0385]

[0386] The results in Table 7 show that the addition of 0.1% Bacillus subtilis G01_spo ferment extract (cell-free supernatant) had a higher percentage increase in TEER value compared to the addition of 0.1% Bacillus subtilis G01, indicating that skin barrier function was further improved compared to the untreated (fermentation extract-free) control sample.

[0387] Example 13

[0388] Knocking out the spoIIE gene from Bacillus subtilis further enhances the extract of Bacillus subtilis ferment. The ability to inhibit the growth of Malassezia

[0389] The ability of Bacillus subtilis G01_spo fermentation extract to inhibit the growth of Malassezia species was evaluated. The Bacillus subtilis G01_spo variant strain (CBS151098) and its parent strain Bacillus subtilis G01 (CBS147469) were grown in a medium containing urea as the primary nitrogen source and glucose as the primary carbon source, supplemented with 1% soybean peptone. Fermentation was carried out in 50 mL centrifuge tubes containing 5 mL of medium. The flasks were placed in a platform shaker at 32°C and a constant speed of 200 rpm for 24 h. Fermentation extract (cell-free supernatant) was obtained by centrifuging the fermentation at 4000 xg for 10 min, followed by filtration of the cell-free supernatant using a 0.22 µm low protein-bound polyethersulfone (PES) membrane filter. The resulting fermentation extract (cell-free supernatant) was stored at 4°C for later use.

[0390] Malassezia furfur strain DSM 6170 was grown in a modified Dixon (mDixon) broth. Each liter of mDixon broth contained 36 g malt extract, 20 g dried ox bile, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. Cultures were produced in 125 mL flat-bottomed shake flasks containing 25 mL of medium. The flasks were placed at 32°C in a platform shaker at a constant speed of 200 rpm for 48 h. The cultures were then diluted with 1% Tween-40 to an OD of 0.42. 600 (1.0 × 10 7 (CFU / mL). Spread 100 µL of the diluted culture onto mDixon agar plates and allow to dry. Each liter of mDixon agar contains 36 g malt extract, 20 g dried ox bile, 15 g Bacto agar, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. Spot 2.5 µL aliquots of Bacillus subtilis G01 and Bacillus subtilis G01_spo fermentation extract onto the center of the Malassezia furfur plate. Incubate the plates overnight at 32°C.

[0391] After overnight incubation, the size of the inhibition zone produced by the fermentation extract in the plate was analyzed. The determination was performed in triplicate, and the average values ​​are shown in Table 10.

[0392] Table 10: Fermentation extracts of Bacillus subtilis G01 and Bacillus subtilis G01_spo Growth inhibition assay of Malassezia using inhibition zone assay

[0393]

[0394] The results in Table 10 show that the Bacillus subtilis G01_spo fermentation extract (cell-free supernatant) had a larger growth inhibition zone compared to the parent strain Bacillus subtilis G01, indicating that the fermentation extract obtained from the Bacillus subtilis spoIIE knockout variant further improved the growth inhibition activity of Malassezia.

[0395] To evaluate the specificity of the activity of the Bacillus subtilis G01_spo ferment extract against other skin microorganisms, Staphylococcus aureus ATCC 27844, Staphylococcus epidermidis ATCC 12228, and Propionibacterium acnes K2 strains were used as target organisms in the inhibition zone assay. The ferment of Bacillus subtilis G01_spo strain was produced as described in Example 4. The strain was grown in SUM (soybean peptone urea micronutrient) medium. Each liter of SUM medium (1.0 X strength) consisted of 10 g soybean peptone, 75 g glucose, 3 ml dipotassium hydrogen phosphate (1 M), 3.6 g urea, and 10 ml of the micronutrient stock solution. Each liter of the micronutrient stock solution contained 1.47 g sodium citrate·2H₂O, 1.47 g CaCl₂·2H₂O, 0.4 g FeSO₄·7H₂O, 0.1 g MnSO₄·H₂O, 0.1 g ZnSO₄·H₂O, 0.05 g CuCl₂·2H₂O, 0.1 g CoCl₂·6H₂O, and 0.1 g Na₂MoO₄·2H₂O. The SUM medium was adjusted to pH 7.3. A 0.5x strength SUM medium was obtained by diluting the medium by half with purified water. The Bacillus subtilis ferment was carried out in 125 ml flat-bottomed shake flasks containing 25 ml of 0.5x strength SUM medium. The flasks were placed in a platform shaker at a constant speed of 200 rpm at 32°C. Overnight fresh seed culture was used as inoculum, with an initial OD of 0.125 at 600 nm. After 24 hours of growth, cell-free supernatant (fermentation extract) was obtained by centrifugation and filtration sterilization.

[0396] In inhibition zone assays against *Malassezia*, direct spotting of 5 µl of a fermentation extract of *Bacillus subtilis* G01_spo did not result in a visible clear zone against either organism. This result indicates that the fermentation extract from *Bacillus subtilis* G01_spo is similar in this respect to its parent strain, *Bacillus subtilis* G01. Both fermentation extract samples exhibited high specificity for inhibiting *Malassezia* growth without affecting the growth of *Staphylococcus hominis*, *Staphylococcus epidermidis*, or *Propionibacterium acnes*.

[0397] To elucidate the molecular weight range of one or more bioactive substances associated with the growth inhibition of *Malassezia* spp. by *Bacillus subtilis* G01-spo strain, a fermentation extract was filtered through a membrane with a 3 kDa cutoff. The effluent was collected and used for inhibition zone determination against *Malassezia furfur* strain DSM 6170. The zone size of the fermentation extract before filtration was 53 mm, and the zone size of the effluent was 50 mm. These results indicate that the less than 3 kDa effluent from the *Bacillus subtilis* fermentation extract contains one or more bioactive agents of less than 3 kDa capable of inhibiting the growth of *Malassezia* spp.

[0398] Example 14

[0399] Knocking out the Bacillus subtilis surfactant operon SRF does not reduce the inhibition of Bacillus subtilis ferment extract. The ability of Malassezia to grow

[0400] Bacillus subtilis strains produce a variety of bioactive metabolites with the ability to inhibit the growth of other microorganisms, such as fungi and bacteria. Among these metabolites, surfactant (srf, SEQ ID NO: 16) is an effective lipopeptide-based biosurfactant (Cheng et al., 2023, AIMS Microbiology, 2023, Vol. 9(2), 195-217).

[0401] The ability of surfactant produced by Bacillus subtilis G01 (CBS147469) to inhibit Malassezia was evaluated.

[0402] A CRISPR-Cas9 plasmid (designated pMY1-6.2) was constructed to delete the srf locus from Bacillus subtilis G01 (CBS147469). The pMY1-6.2 plasmid contains a Streptococcus pyogenes Cas9 endonuclease expression cassette, an editable template insert region (SEQ ID NO: 17), and a kanamycin resistance cassette containing a Bacillus subtilis G01 homologous region and sgRNA (SEQ ID NO: 18). The pMY1-6.2 plasmid was electroporated into Endura™ electrocompetent cells (BioSearch Technologies). The assembled construct was validated via Sanger sequencing. pMY1-6.2 was amplified using rolling circle amplification (RCA) with the GenomiPhi HY Ready-To-Go DNA Amplification Kit (Stenvent).

[0403] Bacillus subtilis G01 (CBS147469) was modified to contain the competent plasmid pBL.comK as described in WO 2019 / 40423, and the srf operon was deleted. Briefly, pBL.comK was transformed into Bacillus subtilis G01 using naturally competent cells and spectinomycin as a resistance marker. The pBL.comK-containing strain was then made competent as described in WO 2021 / 146411 and subsequently transformed with plasmid pMY1-6.2. 200 μL of competent cells was mixed with 50 μL of pMY1-6.2 RCA product and 1 mL of Luria-Bertani (LB) broth and incubated at 30°C and 250 RPM for 1.5 h. The mixture was plated onto LB agar plates containing 20 ppm kanamycin to select cells transformed with the Cas9 plasmid pMY1-6.2. Isolates were screened for deletions of the srf operon using Q5® PCR primers BSsrf-checkdel-F (SEQ ID NO: 19) and BSsrf-checkdel-R (SEQ ID NO: 20). Colonies with srf deletions (Δsrf) produced a PCR product of 2604 bp, while wild-type strains containing the intact srf operon produced PCR products too large to be observed on agarose gels (28,581 bp). Deletion was confirmed by Sanger sequencing. To further confirm that the knockout isolates were not a mixture of srf knockout and wild-type strains, multiplex PCR was performed to confirm individual gene deletions within the srf operon. If the colonies were a mixture, the following band sizes were produced for each gene: srfAA produced 1128 bp, srfAB produced 1325 bp, srfAC produced 854 bp, and srfAD produced 560 bp. Isolates lacking the gene did not produce a band for that gene. The confirmed mutant isolates were stored in glycerol stock solution at -80°C.

[0404] The isolates were eliminated by culturing them in antibiotic-free trypsinized soybean broth (TSB) for three days, subculturing every 24 hours, and then plated onto plain trypsinized soybean agar (TSA). Colonies from the TSA were selected and inoculated onto antibiotic-free LB agar, LB agar containing 20 ppm kanamycin, and LB agar containing 100 ppm spectinomycin. The eliminated isolates grew on plain LB agar and failed to grow on LB agar containing kanamycin or spectinomycin.

[0405] After confirming the srf deletion through genomic analysis, phenotypic analysis of the fermentation broth extract (cell-free supernatant) for *Bacillus subtilis* G01 lacking the srf locus (designated *Bacillus subtilis* G01_srf) was performed using liquid chromatography-ultraviolet (LC-UV) to confirm the loss of surfactant production in *Bacillus subtilis* G01_srf. The *Bacillus subtilis* G01_srf variant and its parent strain *Bacillus subtilis* G01 (CBS147469) were grown in a culture medium using urea as the primary nitrogen source and glucose as the primary carbon source, supplemented with 1% soybean peptone. Fermentation was carried out in 50 mL centrifuge tubes containing 5 mL of culture medium. The flasks were incubated at 32°C in a platform shaker at a constant speed of 200 rpm for 24 h. Fermentation extract (cell-free supernatant) was obtained by centrifuging the fermentation broth at 4000 xg for 10 minutes in a centrifuge, and then the cell-free supernatant was filtered using a 0.22 μm low protein-bound polyethersulfone (PES) membrane filter.

[0406] The fermentation extracts were analyzed by LC-UV, and the samples were quantified using a calibration curve prepared with surfactant standards. The results are shown in Table 11.

[0407] Table 11: Bacillus subtilis G01_srf does not produce surfactants.

[0408]

[0409] The results presented in Table 11 show that the wild-type Bacillus subtilis G01 produced 318.1 mg / L of surfactant, while no surfactant was detected in the Bacillus subtilis G01 Δsrf isolate. Therefore, the deletion of the srf operon removes the ability of Bacillus subtilis G01 to produce surfactant.

[0410] Following phenotypic confirmation of surfactant knockout (Bacillus subtilis G01_srf), the ability of the Bacillus subtilis G01_srf ferment broth extract to inhibit the growth of Malassezia species was evaluated. As previously described, the Bacillus subtilis G01_srf variant and its parent strain, Bacillus subtilis G01 (CBS147469), were grown in culture medium. The resulting ferment broth extract (cell-free supernatant) was stored at 4°C for later use.

[0411] Malassezia furfur strain DSM 6170 was grown in a modified Dixon (mDixon) broth. Each liter of mDixon broth contained 36 g malt extract, 20 g dried ox bile, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. Cultures were produced in 125 mL flat-bottomed shake flasks containing 25 mL of medium. The flasks were placed at 32°C in a platform shaker at a constant speed of 200 rpm for 48 h. The cultures were then diluted with 1% Tween-40 to an OD of 0.42. 600 (1.0 × 10 7 (CFU / mL). Spread 100 μL of the diluted culture onto mDixon agar plates and allow to dry. Each liter of mDixon agar contains 36 g malt extract, 20 g dried ox bile, 15 g Bacto agar, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. Spot 2.5 μL aliquots of Bacillus subtilis G01 wild-type and Bacillus subtilis G01 Δsrf fermentation extract onto the center of the Malassezia furfur plates. Incubate the plates overnight at 32°C.

[0412] After overnight incubation, the size of the inhibition zone produced by the fermentation extract in the plate was analyzed. The determination was performed in triplicate, and the average values ​​are shown in Table 12.

[0413] Table 12: Fermentation extracts of Bacillus subtilis G01 and Bacillus subtilis G01 Δsrf (cell-free) The growth inhibition assay of Malassezia spp. was performed using the inhibition zone assay (Clear liquid).

[0414]

[0415] The results presented in Table 12 show that the inhibition zone of the Bacillus subtilis G01_srf ferment extract (cell-free supernatant) was not reduced when compared with the parent strain Bacillus subtilis G01, indicating that the surfactant produced by Bacillus subtilis G01 does not contribute to the growth inhibition activity of Malassezia.

[0416] Example 15

[0417] Improve skin barrier function using Bacillus subtilis ferment extract fractions with less than 3 kDa.

[0418] The ability of Bacillus subtilis ferment broth extracts with fractions smaller than 3 kilodaltons (kDa) to improve skin barrier function was evaluated. Bacillus subtilis strain G01_spo was grown in a modified SMU medium consisting of 1 g / L soybean peptone, 3 mM KH buffer, 20 g / L glucose, 1.8 g / L urea, 0.735 g / L sodium citrate·2H2O, 0.735 g / L CaCl2·2H2O, 0.2 g / L FeSO4·7H2O, 0.05 g / L MnSO4·H2O, 0.05 g / L ZnSO4·H2O, 0.0025 g / L CuCl2·2H2O, 0.05 g / L CoCl2·6H2O, and 0.05 g / L Na2MoO4·2H2O. After 24 hours of growth, the ferment broth was sterilized by centrifugation and filtration to obtain the ferment broth extract (cell-free supernatant). After centrifugation and filtration sterilization, the ferment extract was then molecularly fractionated by passing it through a series of molecular weight cutoff filters in the following order: 30 kilodaltons (kDa), 10 kDa, and 3 kDa. The ferment extract that did not pass through the filters was retained and referred to as the osmotic residue (R), and the ferment extract that passed through the filters was retained and referred to as the effluent (FT). A fraction of the 3 kDa FT was then concentrated 10-fold using a speedvac. In short, 3 mL of the 3 kDa FT fraction was centrifuged at room temperature under vacuum until a final volume of 300 μL was reached. This fraction was designated 3 kDa FT 10X. The ability of the unconcentrated 3 kDa FT and 3 kDa FT 10X fractions of Bacillus subtilis G01_spo strain to improve skin barrier function was tested using NHEK as performed in Example 12, and the results were compared to an untreated control (without ferment extract). The results are shown in Table 13.

[0419] Table 13. < 3 kDa effluent Bacillus subtilis fermentation extract fractions show increased transepithelial resistivity (TEER).

[0420]

[0421] The results in Table 13 show that, compared with the untreated (fermentation-free extract) control, the addition of less than 3 kDa of the unconcentrated and concentrated forms of Bacillus subtilis G01_spo fermentation extract (cell-free supernatant) resulted in a higher percentage increase in TEER values, indicating improved skin barrier function.

[0422] Example 16

[0423] Knocking out the Bac operon of Bacillus subtilis reduces the inhibition of Marrakech by Bacillus subtilis ferment extract. The ability of Chromophytum to grow

[0424] The ability of the Bacillus subtilis bacilysin operon bac (bac A-bac B-bac C-bac D-bac E-bacF-bac G, SEQ ID NO:21) to produce a bacilysin-type dipeptide for inhibiting Malassezia species was evaluated.

[0425] A CRISPR-Cas9 plasmid (designated pMY1-13.1) was constructed to delete the bac locus from Bacillus subtilis G01 (CBS147469). The pMY1-13.1 plasmid contains a Streptococcus pyogenes Cas9 endonuclease expression cassette, an editable template insert region (SEQ ID NO: 22), and a kanamycin resistance cassette containing a Bacillus subtilis G01 homologous region and sgRNA (SEQ ID NO: 23). The pMY1-13.1 plasmid was electroporated into Endura™ electrocompetent cells (BioSearch Technologies). The assembled construct was validated via Sanger sequencing. pMY1-13.1 was amplified using rolling circle amplification (RCA) with the GenomiPhi HY Ready-To-Go DNA Amplification Kit (Stenvent).

[0426] Bacillus subtilis G01 (CBS147469) was modified to contain the competent plasmid pBL.comK as described in WO 2019 / 40423, and the bac operon was deleted. Briefly, pBL.comK was transformed into Bacillus subtilis G01 using naturally competent cells and spectinomycin as a resistance marker. The pBL.comK-containing strain was then made competent as described in WO 2021 / 146411 and subsequently transformed with plasmid pMY1-13.1. 200 μL of competent cells were mixed with 50 μL of pMY1-13.1 RCA product and 1 mL of Luria-Bertani (LB) broth and incubated at 30°C and 250 RPM for 1.5 h. The mixture was plated onto LB agar plates containing 20 ppm kanamycin to select cells transformed with the Cas9 plasmid pMY1-13.1. Isolates were screened for deletions of the bac operon using Q5® PCR primers BSbac-checkdel-F (SEQ ID NO: 24) and BSbac-checkdel-R (SEQ ID NO: 25). Colonies with the bac deletion (Δbac) produced a 2417 bp PCR product, while wild-type strains containing the intact bac operon produced a 9121 bp PCR product. The deletion was confirmed by Sanger sequencing. Confirmed mutant isolates were stored in glycerol stock solution at -80°C.

[0427] The isolates were eliminated by culturing them in antibiotic-free trypsinized soybean broth (TSB) for three days, subculturing every 24 hours, and then plated onto plain trypsinized soybean agar (TSA). Colonies from the TSA were selected and inoculated onto antibiotic-free LB agar, LB agar containing 20 ppm kanamycin, and LB agar containing 100 ppm spectinomycin. The eliminated isolates grew on plain LB agar and failed to grow on LB agar containing kanamycin or spectinomycin.

[0428] After genotypic confirmation of the bac operon knockout, the ability of the Bacillus subtilis G01 baclysin knockout mutant (hereinafter referred to as Bacillus subtilis G01_bac) to inhibit the growth of Malassezia species was evaluated. The Bacillus subtilis G01_bac variant and its parent strain, Bacillus subtilis G01 (CBS147469), were grown in culture medium. The medium used urea as the primary nitrogen source and glucose as the primary carbon source, supplemented with 1% soybean peptone. Fermentation was carried out in 50 mL centrifuge tubes containing 5 mL of culture medium. The flasks were placed in a platform shaker at 32°C and a constant speed of 200 rpm for 24 h. Fermentation extract (cell-free supernatant) was obtained by centrifuging the fermentation product at 4000 xg for 10 min, followed by filtration of the cell-free supernatant using a 0.22 μm low protein-bound polyethersulfone (PES) membrane filter. The resulting ferment extract (cell-free supernatant) was stored at 4°C for later use.

[0429] Malassezia furfur strain DSM 6170 was grown in a modified Dixon (mDixon) broth. Each liter of mDixon broth contained 36 g malt extract, 20 g dried ox bile, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. Cultures were produced in 125 mL flat-bottomed shake flasks containing 25 mL of medium. The flasks were placed at 32°C in a platform shaker at a constant speed of 200 rpm for 48 h. The cultures were then diluted with 1% Tween-40 to an OD of 0.42. 600 (1.0 × 10 7(CFU / mL). Spread 100 μL of the diluted culture onto mDixon agar plates and allow to dry. Each liter of mDixon agar contains 36 g malt extract, 20 g dried ox bile, 15 g Bacto agar, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. Spot 2.5 μL aliquots of Bacillus subtilis G01 wild-type and Bacillus subtilis G01 Δbac fermentation extract onto the center of the Malassezia furfur plates. Incubate the plates overnight at 32°C.

[0430] After overnight incubation, the size of the inhibition zone produced by the fermentation extract in the plate was analyzed. The determination was performed in triplicate, and the average values ​​are shown in Table 14.

[0431] Table 14: Fermentation extracts of Bacillus subtilis G01 and Bacillus subtilis G01_bac (cell-free supernatant) The growth inhibition assay of Malassezia was performed using the inhibition zone assay.

[0432]

[0433] The results presented in Table 14 show that the Bacillus subtilis G01_bac fermentation extract (cell-free supernatant) did not produce inhibition zones, while the parental Bacillus subtilis G01 strain produced zones of 36 mm. This demonstrates that the absence of the bac operon eliminates the ability of Bacillus subtilis G01 to inhibit the growth of Malassezia, and therefore indicates that this pathway facilitates the ability of Bacillus subtilis G01 to inhibit the growth of Malassezia.

Claims

1. A skin care composition for providing at least one skin care benefit in a subject in need, the composition comprising an effective amount of Bacillus subtilis ferment extract or a fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

2. The skin care composition of claim 1, wherein the Bacillus subtilis ferment extract is selected from the group consisting of: cell-free supernatant obtained from Bacillus subtilis ferment, cell precipitate extract obtained from Bacillus subtilis ferment, whole broth ferment extract obtained from Bacillus subtilis ferment, and any combination thereof.

3. The skin care composition of claim 1, wherein the scalp or skin disorder is selected from the group consisting of: scalp microbiota imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, and any combination thereof.

4. The skin care composition of claim 1, wherein reducing dandruff comprises applying the composition to the skin or scalp of the subject to reduce dandruff.

5. The skin care composition according to any of the preceding claims, wherein the Bacillus subtilis ferment extract or fraction thereof is derived from Bacillus subtilis ferment, wherein the Bacillus subtilis is selected from the group consisting of: Bacillus subtilis strains having a 16S ribosomal RNA sequence (SEQ ID NO: 1) exhibiting at least 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and at most 100% sequence similarity to the 16S ribosomal RNA sequence (SEQ ID NO: 1) of Bacillus subtilis G01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB), and Bacillus subtilis strains having a 16S ribosomal RNA sequence (SEQ ID NO: 1) of Bacillus subtilis PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB), exhibiting at least 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and at most 100% sequence similarity to the 16S ribosomal RNA sequence (SEQ ID NO: 1) of Bacillus subtilis PC01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB), exhibiting at least 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 2) Bacillus subtilis strains exhibiting 16S ribosomal RNA sequences with at least 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and up to 100% sequence similarity, and possessing the 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis N01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149971. 3) Bacillus subtilis strains exhibiting 16S ribosomal RNA sequences with at least 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and up to 100% sequence similarity, and possessing the 16S ribosomal RNA sequence (SEQ ID NO: ) of Bacillus subtilis K01 deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with accession number CBS149972. 7) Bacillus subtilis strains exhibiting 16S ribosomal RNA sequences showing at least 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and up to 100% sequence similarity, and possessing a 16S ribosomal RNA sequence (SEQ ID NO:1) of Bacillus subtilis G01_spo deposited at the Westdick Institute for Fungal Biodiversity (WFDB) with the number CBS151098, showing at least 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, and up to 100% sequence similarity.Bacillus subtilis strains with 16S ribosomal RNA sequences exhibiting 9% to up to 100% sequence similarity, and any combination thereof.

6. The skin care composition as claimed in any of the preceding claims, wherein the skin care composition further comprises one or more anti-dandruff active agents.

7. The skin care composition of claim 1, wherein the composition reduces the growth of Malassezia species without affecting the growth of one or more bacteria selected from the group consisting of: Staphylococcus hominis, Staphylococcus epidermis, Cutibacteria acnes strains, and any combination thereof.

8. The skin care composition of claim 1, wherein the skin barrier strengthening and / or the improvement of skin barrier function and / or the skin moisturizing occur through fatty acid synthesis, keratinization, synthesis of small proline-rich proteins, or any combination thereof.

9. The skin care composition as claimed in any of the preceding claims, wherein the skin care composition further comprises additional compounds selected from the group consisting of: fragrances, excipients, preservatives, pH adjusters, aqueous carriers, alcohol carriers, emollients, curing agents, hydrating agents, emulsifiers, solubilizers, surfactants, thickeners, salts, and any combination thereof.

10. Use of the skin care composition as claimed in any of the preceding claims in a cosmetic skin care product.

11. A skin care product comprising a skin care composition as described in any of the preceding claims and one or more dermatologically or skin care-acceptable components.

12. The skin care product of claim 11, wherein the skin care product comprises at least about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% of the skin care composition by weight relative to the total weight of the skin care product.

13. The skin care product of claim 11, wherein the product is formulated for topical application.

14. The skin care product according to any one of claims 11-13, wherein the skin care product is selected from the group consisting of: lotions, serums, gels, creams, gels, hydrogels, lotions, solid cosmetics, films, patches, shampoos, loose or dense powders, liquid suspensions or solutions, spray solutions and sticks.

15. A method for providing at least one skin care benefit to a subject in need, the method comprising applying topically to the skin or scalp of the subject a skin care composition or skin care product as described in any of the preceding claims.

16. A method for reducing dandruff in a subject, the method comprising topically applying to the subject's scalp a skin care composition or skin care product as described in any of the preceding claims.

17. A method for strengthening the skin barrier of a subject, the method comprising topically applying to the skin or scalp of the subject a skin care composition or skin care product as described in any of the preceding claims.

18. A method for moisturizing the skin of a subject, the method comprising applying topically to the skin or scalp of the subject a skin care composition or skin care product as described in any of the preceding claims.

19. The method of any one of claims 15-18, wherein the Bacillus subtilis ferment extract is selected from the group consisting of: cell-free supernatant obtained from Bacillus subtilis ferment, cell precipitate extract obtained from Bacillus subtilis ferment, whole broth ferment extract obtained from Bacillus subtilis ferment, and any combination thereof.

20. A Bacillus subtilis strain variant ferment, Bacillus subtilis strain variant ferment extract, or fraction of said variant ferment, wherein said Bacillus subtilis strain variant is derived from said parent Bacillus subtilis by knocking out at least one sporulation gene of a parent Bacillus subtilis strain, wherein said variant ferment, said variant ferment extract, or said fraction of said variant ferment has increased activity compared to the activity of a ferment, ferment extract, or fraction of said variant ferment, wherein said activity is selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof.

21. A method for increasing the activity of a Bacillus subtilis strain ferment extract for providing at least one skin care benefit, the method comprising: (a) Providing a parent Bacillus subtilis strain capable of producing an effective amount of a ferment extract for providing at least one skin care benefit selected from the group consisting of: reducing dandruff, reducing scalp or skin barriers, strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof; (b) Modifying the parental Bacillus subtilis strain by knocking out at least one spore-forming gene from the genome of the Bacillus subtilis strain, thereby generating a variant strain that cannot form spores; and, (c) Producing a fermentation extract of the Bacillus subtilis variant strain, wherein the fermentation extract of the Bacillus subtilis variant strain has increased activity when compared with the activity of the fermentation extract of the parental Bacillus subtilis strain, for providing in subjects in need at least one skin care benefit selected from the group consisting of: reduced dandruff, reduced scalp or skin barrier, strengthened skin barrier, improved skin barrier function, skin hydration, and any combination thereof.

Images