Fermentate extracts for providing skin care benefits and methods thereof

Abstract

The present disclosure is directed towards 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 is directed towards methods and compositions comprising a Bacillus subtilis fermentate, Bacillus subtilis fermentate extract, or fraction thereof, for reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

Description

[0001] TITLE

[0002] FERMENTATE EXTRACTS FOR PROVIDING SKIN CARE BENEFITS AND METHODS THEREOF

[0003] 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 are incorporated by reference herein in their entireties.

[0004] FIELD OF THE DISCLOSURE

[0005] The present disclosure is directed towards 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 is directed towards methods and compositions comprising a Bacillus subtilis fermentate, Bacillus subtilis fermentate extract, or fraction thereof, for reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0006] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0007] The content of the sequence listing electronically submitted with the application as an XML file (Name: 20240617_NB42207PCT_SequenceListing.xml; Size: 72,375 bytes; Created: June 17, 2024) forms part of the application and is hereby incorporated herein by reference in its entirety.

[0008] BACKGROUND

[0009] The skin functions as a barrier protecting the organism from drying out as well as protecting the organism against the penetration of external, often harmful, substances. The skin barrier is important to human life as it physically protects from external threats such as infectious agents, chemicals, systemic toxicity and allergens, and internally helps the skin to maintain homeostasis and protects from enhanced loss of water from the body (Skin barrier function, 2016, T. Agner, editor, Karger, Basel, Switzerland, 164 pages, ISBN 978-3-318-05585-6). The human skin consists of two main layers of cells, epidermis and dermis. The epidermis constitutes the outermost layer of the skin and is mainly formed of terminally differentiated keratinocytes and lipids, living dividing keratinocytes located beneath the terminally differentiated ones. The outer layer of the epidermis (Stratum corneum or horny layer) is the part which is in contact with the environment and the particular structure of the homy layer protects the skin as well as stabilizes its own flexibility by binding a defined amount of water (P. M. Elias, Structure and Function of the Stratum Corneum Permeability Barrier, Drug Dev. Res. 13, 1988, 97-105). The skin barrier function is greatly dependent on the structure and composition of the stratum corneum, which is made up of flattened anucleated cells surrounded by highly organized and continuous lipid matrix (Skin barrier function, 2016, T. Agner, editor, Karger, Basel, Switzerland, 164 pages, ISBN 978-3-318-05585-6). The main function of the epidermis is to form permeability barrier against environmental challenges, such as UV radiation, heat, chemicals, pollution, and pathogens, such as bacteria, fungi, parasites, and viruses. It also protects the body from uncontrolled water evaporation from inside out, maintaining the hydration balance and skin metabolism.

[0010] The stratified epithelium of the skin is comprised of multiple layers of keratinocytes in various states of differentiation. The layers, from the most upper layer to the bottom layer, are commonly referred to as stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum basale. Collectively, these layers impart multiple functionalities of the skin with providing a physical barrier to the external environment and microbes being the primary function (Hogan et al., 2012, Journal of Allergy 2012:901940:1 -7). A defective skin barrier is widely postulated to contribute to many 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 T. C., et. al., 2019, Experimental Dermatology 28(9):991 - 1001 ) as well as dry skin and skin irritation.

[0011] As keratinocytes differentiate, the cells express a multitude of proteins, enzymes and lipids that participate in forming the stratum corneum, which is the first line barrier of the skin. Integral features of the stratum corneum that contribute to the integrity of the skin’s barrier are cross-linked structural proteins, lipids and tightly linked cell-cell attachments. Genetic mutations resulting in impaired production of these components are implicated in many skin disorders demonstrating that all barrier components are important for maintaining skin health (Eckert, R. L. 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 J.Y.W. and J. A. McGrath, 2021 , British Journal of Dermatology 184(4): 596-605).

[0012] Tightly adjoined keratinocytes in the stratum corneum is one feature that maintains skin barrier integrity thereby limiting water loss and preventing 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, Cold Spring Harbor Perspectives in Medicine 4(11 )). Expression of a series of transmembrane proteins is one strategy keratinocytes use to link together. Three transmembrane systems important for ensuring neighboring keratinocytes are tightly linked together are desmosomes, cadherins (Cdh) and protocadherins (Pcdh). Desmosomes, intracellular adherence junctions that link intermediate filaments of neighboring cells, are comprised of key proteins such as desmogleins (Dsg), desmocollins (Dsc) and desmoplankin (DP). Disruption of desmosome formation can lead to loss of skin barrier integrity (Lee J.Y.W. and J. A. McGrath, 2021 , British Journal of Dermatology 184(4): 596-605; Johnson J. et al., 2014, Cold Spring Harbor Perspectives in Medicine 4:(11 )). Cadherins and the cadherin subfamily protocadherins, like desmosomes, are key intracellular adherence junctions that link actin filaments of neighboring cells. Both represent large classes of proteins, with protocadherins further distinguished by three sub-classes: alpha, beta and gamma. Disruption of cadherin and protocadherin function results in loss of epithelial barrier integrity and can result in development of disease (Brandner J., et al. , 2010, The Open Dermatology Journal 4:14-20; Pancho A., et. al., 2020, Frontiers in Molecular Neuroscience 13:117; Tinkle C. L., et. al., 2008, PNAS 105(40): 15405-15410).

[0013] Keratinocytes in the stratum comeum, through the differentiation process, form a specialized membrane termed the cornified envelope. The cornified envelope is primarily comprised of crosslinked structural proteins and lipids and contribute to maintaining the integrity of the skin barrier. Important proteins that impart functionality to the cornified envelope include filaggrin, involucrin loricrin, late envelope proteins (LCE) and sciellin (SCEL). Enzymes are required to crosslink proteins such as involucrin and loricrin, of which transglutimanses (TGM) are the most prevalent. Ceramides and long chain fatty acids are amongst the most abundant lipids in the stratum corneum and help provide a hydrophobic barrier in order to maintain water and moisture homeostasis. Important to synthesizing these lipids are a class of enzymes termed ceramide synthases (CERS) and elongases of very long chain fatty acids (ELOVL). Ceramides require the addition of a sphingolipid to a fatty acid and therefore enzymes such as FA2H, fatty acid 2- hydroxylase, are required for the generation of sphingolipids (Proksch E., et al., 2008, Experimental Dermatology 17: 1063-1072; Uchida Y., et. al., 2007, Journal of Biological Chemistry 282(18): 13211 -13219)

[0014] An important function of the skin barrier is to maintain moisturization, which involves careful balancing of water content (Proksch E., et al., 2008, Experimental Dermatology 17: 1063-1072). In general, the skin barrier prevents excessive water loss and water absorption to ensure the skin remains moist and does not dry out. One key component of the skin epidermal layer that maintains moisturization is hyaluronic acid (HA)(Papakonstantinou E., et al., 2012, DermatoEndicrinolgy 4(3):253-258). In order to be completely effective, HA is cross-linked, which is mediated by enzymes such as TNFAIP6 (Evrard C., et. al., 2021 , JID Innovations 1 :100054). Disruption of the physical barrier, such caused by an abrasion or inflammatory diseases of the skin including but limited to eczema, psoriasis and atopic dermatitis, can disrupt the balance of water regulation. Therefore, upon disruption of the physical skin barrier, proper function of cellular processes necessary to return the skin barrier back to its intact form are imperative. Chronic exposure of the under-laying layers of the epidermis and dermis, as exhibited in conditions such as eczema, psoriasis, atopic dermatitis, contact dermatitis, burns and ulcers due to complications from diabetes, vascular inflammation and ischemic injury, can result in dryness and chronic inflammation due to persistent exposure from microbial pathogens and environmental pollutants (Proksch E., et al., 2008, Experimental Dermatology 17:1063-1072). A common method to measure skin barrier integrity and by association skin moisturization is measuring transepidermal water loss (TEWL). TEWL measurements indicate the amount of water evaporation at the surface of the skin. Higher TEWL values are indicative of higher water loss and therefore associated with dryer skin (Proksch E., et al., 2008, Experimental Dermatology 17:1063-1072). Measuring electrical resistance of the skin barrier is another method of assessing skin barrier integrity. Higher resistance values are correlated to better barrier integrity, whereas lower resistance values are correlated to poorer barrier integrity. TEWL values are known to coincide with electrical resistance measurements in laboratory / research settings (Guth K., et. al., 2015, Toxicology In Vitro 29:113-123). Thus, lower TEWL values can be reflected by higher electrical resistance values and therefore indicate an intact barrier leading to minimal water loss.

[0015] Current treatments for most skin disorders directly target either inflammation or implicated microbe, which when either component is inhibited allows the skin to recover to form an intact barrier. For instance, inhibition of cytokines using biologicals such as monoclonal antibodies in skin disorders such as psoriasis and atopic dermatitis allows expression of filaggrin and other barrier components to normalize (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). Particular to dandruff, anti-fungal components in anti-dandruff shampoos such as zinc pyrithione (ZPT) and octipirox directly control the out-growth of multiple Malassezia species. Reducing the out-growth of Malassezia reduces scalp inflammation and subsequently leads to the restoration of the skin barrier (Warner, R. R., et. al., 2001 , Journal of the American Academy of Dermatology 45(6): 897- 903). Other treatments such as salicylic acid and coal tar help clear dead skin (Ranganathan, S. and T. Mukhopadhyay, 2010, Indian Journal of Dermatology 55(2): 130-134).

[0016] When Senkyunolide A (SENKY), a phthalide extracted from celery seed was applied to the scalp of individuals with dandruff, a reduced dandruff score was observed, as well as reduced flaking and improved trans-epidermal water loss (TEWL). Mechanistically, SENKY increases the expression of various skin barrier components including, but not limited to, loricrin, filaggrin, SPRR2B and LCE3B. This study indicates that improving the skin barrier can help with reducing the occurrence of dandruff (Mondon, P. et. al, 2017, International Journal of Cosmetic Science, 39: 617-621 ).

[0017] The skin is also home to a diverse population of microbes, the majority of which are commensal (nonpathogenic permanent residents) or transient (temporary residents) organisms. In pathogenic interactions, only the microbe benefits, while the host is eventually harmed. Many skin pathogens can be typically found living on the skin as commensal organisms, but microbial dysbiosis (or microbial imbalance), host genetic variation, and immune status may drive the transition from commensal to pathogen (Findley, K. and Grice, E. A., The Skin Microbiome: A Focus on Pathogens and Their Association with Skin Disease. PLoS Pathog. 2014, 10).

[0018] The epidermis constitutes the outermost region of the skin tissue and as such forms the actual protective sheath against the environment. The outer layer of the epidermis (Stratum corneum or Horny layer) is the part which is in contact with the environment and the particular structure of the horny layer protects the skin as well as stabilizes its own flexibility by binding a defined amount of water (P. M. Elias, Drug Dev. Res. 13, 1988, 97-105).

[0019] Spatially, the skin microbiota may extend to subepidermal compartments (Nakatsuji, T et al., The Microbiome Extends to Subepidermal Compartments of Normal Skin. Nat. Commun. 2013, 4). Regions such as the face, chest, and back, areas with a high density of sebaceous glands, promote growth of lipophilic microorganisms such as Propionibacterium and Malassezia. Skin is a unique environment where microbes often exist as biofilms (Brandwein, et al., 2016. NPJ Biofilms Microbiomes 2:3). The biofilms can form on the epithelial surfaces of the skin or inside the follicles. In addition to cells, a biofilm consists of extracellular components such as exopolysaccharides, proteins, and DNA. This complex structure can be a physical and chemical barrier for certain compounds. But more importantly, the physiology of the microbes in the state of biofilm is very different than those in planktonic state. This is especially true for their ability to counter environmental stress and to resist various antimicrobial treatments (Koo, et al., Nature Reviews Microbiology 15:740-755, 2017).

[0020] Yeast Malassezia species isolated from both healthy and unhealthy skin have been shown to form biofilms in vitro (Angiolella, et al. 2020, Med Mycol. 0:1- 7). These isolates of Malassezia globosa (M. globosa) can be highly adherent and / or hydrophobic as well as biofilm producers. Malassezia species in the form of biofilm have been shown to have a significant decrease in their susceptibility to antifungal agents (Figueredo, et al., 2016, Medical Mycology 8:863-867, 2013; Bumroogthai, et al., Medical Mycology 54:544-549). Biofilm adherence and hydrophobicity was suggested as virulence factor for Malassezia (Allen, et al., 2018, J. of Clinical & Experimental Dermatology Research 6:311 , 2015; Angiolella, et al., Medical Mycology 56:110-116). Thus, strategies to remove Malassezia biofilm can be beneficial to treat various skin conditions caused by this group of organisms.

[0021] Malassezia is a predominant fungus of the skin microbiota and found on virtually everybody's scalp and implicated in the most common skin disorders such as, but not limiting to, seborrheic dermatitis, dandruff, and tinea versicolor. Dandruff is the common term for seborrhea of the scalp. It is mainly associated with Malassezia species such as Malassezia restricta (M. restricta) and Malassezia globosa (M. globosa) and has a very high prevalence of nearly 50% of the population (Schommer, N. N.; Gallo, R. L., Structure and Function of the Human Skin Microbiome. Trends Microbiol. 2013, 21 , 660-668). Improvements in the disorder can be achieved by therapeutic application of antifungal, but not antibacterial agents. The mechanisms underlying pathogenicity are incompletely understood. Impaired skin barrier function facilitates the course of the disorder (Harding, C. Ret 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).

[0022] Malassezia species do not have fatty acid synthase, so they have to rely on sebum lipids for carbon source. They also lack delta 2,3-enoyl-CoA isomerase for efficient unsaturated FA (e.g. oleate) utilization. Malassezia species feeds on sebum fat (by secreting a lipase or lipases that splits triglycerides into irritant fatty acids), and as sebum fat is broken down, free fatty acids (such as oleic acid) are released as by-product. Many people are sensitive to free fatty acids as they can induce hyperproliferation and scaling, or induce the release of arachidonic acid, which is also involved in inflammation, and their scalp responds by becoming irritated. In response to the irritation, the scalp starts to become inflamed, red, and itchy, and the body shed skin cells faster than usual, in attempt to shed the irritant. The shedding of skin causes visible flakes to appear on the scalp, which is dandruff.

[0023] Human skin is colonized by a diverse microbiota (Byrd, A., Belkaid, Y. & Segre, J. The human skin microbiome. Nat Rev Microbiol 16, 143-155; 2018). Staphylococcus hominis, Staphylococcus epidermis and Cutibacteria acnes strains (formerly known as Propioni bacterium) are known commensal species.

[0024] There remains a need to find methods and skin care compositions for reducing dandruff condition of the scalp and for reducing a scalp or skin disorder. Furthermore, there remains a need to find methods and skin care compositions for skin barrier strengthening, improving skin barrier function and skin moisturization of a skin or scalp.

[0025] SUMMARY

[0026] The present disclosure is directed towards 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 is directed towards methods and compositions comprising a Bacillus subtilis fermentate, Bacillus subtilis fermentate extract, or fraction thereof, for reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0027] The inventors have unexpectedly observed that a fermentate extract (cell free supernatant) of Bacillus subtilis (such as but not limiting to Bacillus subtilis N01 , Bacillus subtilis PC01 , Bacillus subtilis G01 , Bacillus subtilis G01_spo, and Bacillus subtilis K01 ) reduces the growth of Malassezia species. The inventors have also unexpectedly observed that a fermentate extract (cell free supernatant) of Bacillus subtilis (such as, but not limiting to, B. subtilis G01 and B. subtilis G01_spo) not only reduces the growth of Malassezia species but does so without affecting the growth of the commensal bacteria Staphylococcus hominis, Staphylococcus epidermis and Cutibacteria acnes strains. Furthermore, the inventors have unexpectedly observed that a fermentate extract (cell free supernatant) of Bacillus subtilis (such as but not limiting to Bacillus subtilis G01 , Bacillus subtilis G01_spo and Bacillus subtilis N01 ) increases Transepidermal Electrical Resistance (TEER), increases expression of genes involved in fatty acid synthesis, cornification, and small proline rich protein synthesis, which is an indication of improved skin barrier function and strengthening of the skin barrier and therefore has suitable use in skin moisturization, strengthening of the skin barrier, improving skin barrier function, reducing dandruff, and reducing skin and scalp disorders.

[0028] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0029] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein the Bacillus subtilis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus subtilis fermentate, a whole broth fermentate extract obtained from a Bacillus subtilis fermentate, and any one combination thereof.

[0030] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein the scalp or skin disorder is selected from the group consisting of an unbalanced ecoflora of the scalp, discomfort of the scalp, tinea versicolor, dry skin, irritated skin, and any one combination thereof.

[0031] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein the composition reduces growth of Malassezia species without affecting growth of Staphylococcus hominis, Staphylococcus epidermis and Cutibacteria acnes strains.

[0032] In one embodiment, the composition is a Bacillus subtilis strain variant fermentate, Bacillus subtilis strain variant fermentate extract, or fraction of said variant fermentate, of a B. subtilis strain variant, wherein said Bacillus subtilis strain variant is derived from a parental B. subtilis by knocking out a sporulation gene of said parental B. subtilis strain, wherein said variant fermentate, said variant fermentate extract, or said fraction of said variant fermentate, has increased activity when compared to the activity of a fermentate, a fermentate extract, or fraction thereof derived from said parental B. subtilis strain, wherein said activity is selected from the group consisting of reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0033] In one embodiment, the skin care product is skin care product comprising a skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0034] In one embodiment, the method is a method for providing at least one skin care benefit to a subject in need thereof, the method comprising topically administering a skin care composition or skin care product comprising said composition to the skin or scalp of said subject, wherein said composition comprises an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0035] DETAILED DESCRIPTION

[0036] The features and advantages of the present disclosure will be more readily understood, by those of ordinary skill in the art from reading the following detailed description. It is to be appreciated that certain features of the disclosure, which are, for clarity, described above and below in the context of separate embodiments, may also be provided in combination in a single element. Conversely, various features of the disclosure that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination. It will be understood that in the following, embodiments referred to in relation to one broad aspect of the invention are equally applicable to each of the other broad aspects of the present invention described above. It will be further understood that, unless the context dictates otherwise, the embodiments described below may be combined.

[0037] Microorganisms, Fermentates, and Fermentate Extracts

[0038] As used herein, "microorganism" or “microbe” refers to a bacterium, a fungus, a virus, a protozoan, archaea, and other microbes or microscopic organisms.

[0039] In some embodiments, the microorganism(s) suitable for use in the present invention can be subjected to treatments that render them non-replicating, for example, exposure to heat, desiccation, y-irradiation, or UV-irradiation. A nonreplicating microorganism(s) suitable for use in the present invention can be a dead cell or a living cell that has been rendered incapable of cell division. A nonreplicating microorganism(s) suitable for use in the present invention can be an intact cell or a cell that has undergone partial or complete lysis. In some embodiments, the non-replicating cells can include a mixture of intact and lysed cells.

[0040] The microorganism(s) suitable for use in the present invention may be included in a composition according to the invention in live, semi-active or inactivated or dead form. For the purposes of the invention, an “inactivated” or “dead” microorganism is a microorganism that is no longer capable of forming colonies in cultures. The dead or inactivated microorganisms may have intact or broken cell membranes. The dead or inactivated microorganisms may be obtained via any method known to those skilled in the art.

[0041] In one aspect the microorganism suitable for use in the present invention includes Bacillus subtilis.

[0042] In some aspects the microorganism suitable for use in the present invention includes a Bacillus subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of Bacillus subtilis MDI_063_G01 (referred to herein as Bacillus subtilis G01 ) deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a bacterial strain having a 16S ribosomal RNA sequence displaying 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%sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a bacterial strain having a 16S ribosomal RNA sequence displaying 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%sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a bacterial strain having a 16S ribosomal RNA sequence displaying 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%sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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%sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0043] The 16S ribosomal RNA sequence of B. subtilis G01 , (SEQ ID NO:1 ) is as follows: AUCGGAGAGUUUGAUCCUGGCUCAGGACGAACGCUGGCGGCGUGCCUAAU ACAUGCAAGUCGAGCGGACAGAUGGGAGCUUGCUCCCUGAUGUUAGCGGC GGACGGGUGAGUAACACGUGGGUAACCUGCCUGUAAGACUGGGAUAACUC CGGGAAACCGGGGCUAAUACCGGAUGGUUGUUUGAACCGCAUGGUUCAAA CAUAAAAGGUGGCUUCGGCUACCACUUACAGAUGGACCCGCGGCGCAUUA GCUAGUUGGUGAGGUAACGGCUCACCAAGGCGACGAUGCGUAGCCGACCU GAGAGGGUGAUCGGCCACACUGGGACUGAGACACGGCCCAGACUCCUACG GGAGGCAGCAGUAGGGAAUCUUCCGCAAUGGACGAAAGUCUGACGGAGCA ACGCCGCGUGAGUGAUGAAGGUUUUCGGAUCGUAAAGCUCUGUUGUUAGG GAAGAACAAGUACCGUUCGAAUAGGGCGGUACCUUGACGGUACCUAACCAG AAAGCCACGGCUAACUACGUGCCAGCAGCCGCGGUAAUACGUAGGUGGCAA GCGUUGUCCGGAAUUAUUGGGCGUAAAGGGCUCGCAGGCGGUUUCUUAAG UCUGAUGUGAAAGCCCCCGGCUCAACCGGGGAGGGUCAUUGGAAACUGGG GAACUUGAGUGCAGAAGAGGAGAGUGGAAUUCCACGUGUAGCGGUGAAAU GCGUAGAGAUGUGGAGGAACACCAGUGGCGAAGGCGACUCUCUGGUCUGU AACUGACGCUGAGGAGCGAAAGCGUGGGGAGCGAACAGGAUUAGAUACCC UGGUAGUCCACGCCGUAAACGAUGAGUGCUAAGUGUUAGGGGGUUUCCGC CCCUUAGUGCUGCAGCUAACGCAUUAAGCACUCCGCCUGGGGAGUACGGU CGCAAGACUGAAACUCAAAGGAAUUGACGGGGGCCCGCACAAGCGGUGGA GCAUGUGGUUUAAUUCGAAGCAACGCGAAGAACCUUACCAGGUCUUGACAU CCUCUGACAAUCCUAGAGAUAGGACGUCCCCUUCGGGGGCAGAGUGACAG GUGGUGCAUGGUUGUCGUCAGCUCGUGUCGUGAGAUGUUGGGUUAAGUCC CGCAACGAGCGCAACCCUUGAUCUUAGUUGCCAGCAUUCAGUUGGGCACU CUAAGGUGACUGCCGGUGACAAACCGGAGGAAGGUGGGGAUGACGUCAAA UCAUCAUGCCCCUUAUGACCUGGGCUACACACGUGCUACAAUGGACAGAAC AAAGGGCAGCGAAACCGCGAGGUUAAGCCAAUCCCACAAAUCUGUUCUCAG UUCGGAUCGCAGUCUGCAACUCGACUGCGUGAAGCUGGAAUCGCUAGUAA UCGCGGAUCAGCAUGCCGCGGUGAAUACGUUCCCGGGCCUUGUACACACC GCCCGUCACACCACGAGAGUUUGUAACACCCGAAGUCGGUGAGGUAACCUU UUAGGAGCCAGCCGCCGAAGGUGGGACAGAUGAUUGGGGUGAAGUCGUAA CAAGGUAGCCGUAUCGGAAGGUGCGGCUGGAUCACCUCCUUU

[0044] The 16S ribosomal RNA sequence of B. subtilis PC01 , (SEQ ID NO:2) is as follows: AUCGGAGAGUUUGAUCCUGGCUCAGGACGAACGCUGGCGGCGUGCCUAAU

[0045] ACAUGCAAGUCGAGCGGACAGAUGGGAGCUUGCUCCCUGAUGUUAGCGGC

[0046] GGACGGGUGAGUAACACGUGGGUAACCUGCCUGUAAGACUGGGAUAACUC

[0047] CGGGAAACCGGGGCUAAUACCGGAUGCUUGUUUGAACCGCAUGGUUCAAA

[0048] CAUAAAAGGUGGCUUCGGCUACCACUUACAGAUGGACCCGCGGCGCAUUA

[0049] GUUAGUUGGUGAGGUAACGGCUCACCAAGGCAACGAUGCGUAGCCGACCU

[0050] GAGAGGGUGAUCGGCCACACUGGGACUGAGACACGGCCCAGACUCCUACG

[0051] GGAGGCAGCAGUAGGGAAUCUUCCGCAAUGGACGAAAGUCUGACGGAGCA

[0052] ACGCCGCGUGAGUGAUGAAGGUUUUCGGAUCGUAAAGCUCUGUUGUUAGG

[0053] GAAGAACAAGUACCGUUCGAAUAGGGCGGUACCUUGACGGUACCUAACCAG

[0054] AAAGCCACGGCUAACUACGUGCCAGCAGCCGCGGUAAUACGUAGGUGGCAA

[0055] GCGUUGUCCGGAAUUAUUGGGCGUAAAGGGCUCGCAGGCGGUUUCUUAAG

[0056] UCUGAUGUGAAAGCCCCCGGCUCAACCGGGGAGGGUCAUUGGAAACUGGG

[0057] GAACUUGAGUGCAGAAGAGGAGAGUGGAAUUCCACGUGUAGCGGUGAAAU

[0058] GCGUAGAGAUGUGGAGGAACACCAGUGGCGAAGGCGACUCUCUGGUCUGU

[0059] AACUGACGCUGAGGAGCGAAAGCGUGGGGAGCGAACAGGAUUAGAUACCC

[0060] UGGUAGUCCACGCCGUAAACGAUGAGUGCUAAGUGUUAGGGGGUUUCCGC

[0061] CCCUUAGUGCUGCAGCUAACGCAUUAAGCACUCCGCCUGGGGAGUACGGU

[0062] CGCAAGACUGAAACUCAAAGGAAUUGACGGGGGCCCGCACAAGCGGUGGA

[0063] GCAUGUGGUUUAAUUCGAAGCAACGCGAAGAACCUUACCAGGUCUUGACAU

[0064] CCUCUGACAAUCCUAGAGAUAGGACGUCCCCUUCGGGGGCAGAGUGACAG

[0065] GUGGUGCAUGGUUGUCGUCAGCUCGUGUCGUGAGAUGUUGGGUUAAGUCC

[0066] CGCAACGAGCGCAACCCUUGAUCUUAGUUGCCAGCAUUCAGUUGGGCACU

[0067] CUAAGGUGACUGCCGGUGACAAACCGGAGGAAGGUGGGGAUGACGUCAAA

[0068] UCAUCAUGCCCCUUAUGACCUGGGCUACACACGUGCUACAAUGGACAGAAC

[0069] AAAGGGCAGCGAAACCGCGAGGUUAAGCCAAUCCCACAAAUCUGUUCUCAG

[0070] UUCGGAUCGCAGUCUGCAACUCGACUGCGUGAAGCUGGAAUCGCUAGUAA

[0071] UCGCGGAUCAGCAUGCCGCGGUGAAUACGUUCCCGGGCCUUGUACACACC

[0072] GCCCGUCACACCACGAGAGUUUGUAACACCCGAAGUCGGUGAGGUAACCUU

[0073] UUAGGAGCCAGCCGCCGAAGGUGGGACAGAUGAUUGGGGUGAAGUCGUAA

[0074] CAAGGUAGCCGUAUCGGAAGGUGCGGCUGGAUCACCUCCUUU The 16S ribosomal RNA sequence of B. subtilis N01 , (SEQ ID NO:3) is as follows:

[0075] CGAUGCGUAGCCGACCUGAGAGGGUGAUCGGCCACACUGGGACUGAGACA

[0076] CGGCCCAGACUCCUACGGGAGGCAGCAGUAGGGAAUCUUCCGCAAUGGAC

[0077] GAAAGUCUGACGGAGCAACGCCGCGUGAGUGAUGAAGGUUUUCGGAUCGU

[0078] AAAGCUCUGUUGUUAGGGAAGAACAAGUACCGUUCGAAUAGGGCGGUACCU UGACGGUACCUAACCAGAAAGCCACGGCUAACUACGUGCCAGCAGCCGCGG UAAUACGUAGGUGGCAAGCGUUGUCCGGAAUUAUUGGGCGUAAAGGGCUC GCAGGCGGUUUCUUAAGUCUGAUGUGAAAGCCCCCGGCUCAACCGGGGAG GGUCAUUGGAAACUGGGGAACUUGAGUGCAGAAGAGGAGAGUGGAAUUCC ACGUGUAGCGGUGAAAUGCGUAGAGAUGUGGAGGAACACCAGUGGCGAAG GCGACUCUCUGGUCUGUAACUGACGCUGAGGAGCGAAAGCGUGGGGAGCG AACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGAGUGCUAAGU GUUAGGGGGUUUCCGCCCCUUAGUGCUGCAGCUAACGCAUUAAGCACUCC GCCUGGGGAGUACGGUCGCAAGACUGAAACUCAAAGGAAUUGACGGGGGC CCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAAGCAACGCGAAGAACCU UACCAGGUCUUGACAUCCUCUGACAAUCCUAGAGAUAGGACGUCCCCUUCG GGGGCAGAGUGACAGGUGGUGCAUGGUUGUCGUCAGCUCGUGUCGUGAG AUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUGAUCUUAGUUGCCAG CAUUCAGUUGGGCACUCUAAGGUGACUGCCGGUGACAAACCGGAGGAAGG UGGGGAUGACGUCAAAUCAUCAUGCCCCUUAUGACCUGGGCUACACACGU GCUACAAUGGACAGAACAAAGGGCAGCGAAACCGCGAGGUUAAGCCAAUCC CACAAAUCUGUUCUCAGUUCGGAUCGCAGUCUGCAACUCGACUGCGUGAAG CUGGAAUCGCUAGUAAUCGCGGAUCAGCAUGCCGCGGUGAAUACGUUCCC GGGCCUUGUACACACCGCCCGUCACACCACGAGAGUUUGUAACACCCGAAG UCGGUGAGGUAACCUUUUAGGAGCCAGCCGCCGAAGGUGGGACAGAUGAU UGGGGUGAAGUCGUAACAAGGUAGCCGUAUCGGAAGGUGCGGCUGGAUCA CCUCCUUU

[0079] The 16S ribosomal RNA sequence of B. subtilis K01 , (SEQ ID NO:7) is as follows: AUCGGAGAGUUUGAUCCUGGCUCAGGACGAACGCUGGCGGCGUGCC

[0080] UAAUACAUGCAAGUCGAGCGGACAGAUGGGAGCUUGCUCCCUGAUGUUAG

[0081] CGGCGGACGGGUGAGUAACACGUGGGUAACCUGCCUGUAAGACUGGGAUA

[0082] ACUCCGGGAAACCGGGGCUAAUACCGGAUGGUUGUUUGAACCGCAUGGUU

[0083] CAAACAUAAAAGGUGGCUUCGGCUACCACUUACAGAUGGACCCGCGGCGCA

[0084] UUAGCUAGUUGGUGAGGUAACGGCUCACCAAGGCGACGAUGCGUAGCCGA

[0085] CCUGAGAGGGUGAUCGGCCACACUGGGACUGAGACACGGCCCAGACUCCU

[0086] ACGGGAGGCAGCAGUAGGGAAUCUUCCGCAAUGGACGAAAGUCUGACGGA

[0087] GCAACGCCGCGUGAGUGAUGAAGGUUUUCGGAUCGUAAAGCUCUGUUGUU

[0088] AGGGAAGAACAAGUACCGUUCGAAUAGGGCGGUACCUUGACGGUACCUAAC

[0089] CAGAAAGCCACGGCUAACUACGUGCCAGCAGCCGCGGUAAUACGUAGGUG

[0090] GCAAGCGUUGUCCGGAAUUAUUGGGCGUAAAGGGCUCGCAGGCGGUUUCU

[0091] UAAGUCUGAUGUGAAAGCCCCCGGCUCAACCGGGGAGGGUCAUUGGAAAC

[0092] UGGGGAACUUGAGUGCAGAAGAGGAGAGUGGAAUUCCACGUGUAGCGGUG

[0093] AAAUGCGUAGAGAUGUGGAGGAACACCAGUGGCGAAGGCGACUCUCUGGU

[0094] CUGUAACUGACGCUGAGGAGCGAAAGCGUGGGGAGCGAACAGGAUUAGAU

[0095] ACCCUGGUAGUCCACGCCGUAAACGAUGAGUGCUAAGUGUUAGGGGGUUU

[0096] CCGCCCCUUAGUGCUGCAGCUAACGCAUUAAGCACUCCGCCUGGGGAGUA

[0097] CGGUCGCAAGACUGAAACUCAAAGGAAUUGACGGGGGCCCGCACAAGCGG

[0098] UGGAGCAUGUGGUUUAAUUCGAAGCAACGCGAAGAACCUUACCAGGUCUUG

[0099] ACAUCCUCUGACAAUCCUAGAGAUAGGACGUCCCCUUCGGGGGCAGAGUG

[0100] ACAGGUGGUGCAUGGUUGUCGUCAGCUCGUGUCGUGAGAUGUUGGGUUAA

[0101] GUCCCGCAACGAGCGCAACCCUUGAUCUUAGUUGCCAGCAUUCAGUUGGG

[0102] CACUCUAAGGUGACUGCCGGUGACAAACCGGAGGAAGGUGGGGAUGACGU

[0103] CAAAUCAUCAUGCCCCUUAUGACCUGGGCUACACACGUGCUACAAUGGACA

[0104] GAACAAAGGGCAGCGAAACCGCGAGGUUAAGCCAAUCCCACAAAUCUGUUC

[0105] UCAGUUCGGAUCGCAGUCUGCAACUCGACUGCGUGAAGCUGGAAUCGCUA

[0106] GUAAUCGCGGAUCAGCAUGCCGCGGUGAAUACGUUCCCGGGCCUUGUACA

[0107] CACCGCCCGUCACACCACGAGAGUUUGUAACACCCGAAGUCGGUGAGGUAA

[0108] CCUUUUAGGAGCCAGCCGCCGAAGGUGGGACAGAUGAUUGGGGUGAAGUC

[0109] GUAACAAGGUAGCCGUAUCGGAAGGUGCGGCUGGAUCACCUCCUUU In one aspect of the invention, fermentates are provided.

[0110] As used herein, the term "fermentate" is to be understood as a composition (complex mixture) produced by propagating living microorganisms (microbial strains) in a nutrient medium. The fermentate may include a cellular mass component from said microorganisms, unspent media components, and metabolites (i.e., unused substrates and / or fermentation end-products). As used herein, a “cellular mass component” refers to any mixture of proteins, lipids (i.e., membranes), carbohydrates, exopolysaccharides, metabolites, etc. from the propagated microorganism. For example, as a microorganism grows it produces new cells that generally include additional cellular mass such as, without limitation, cell membranes, nucleic acids (i.e., DNA and / or RNA) internal subcellular structures, polysaccharides, and proteins (i.e., membrane-bound, secreted, and / or intracellular).

[0111] Fermentates for use in the present invention include fermentates from the microorganism Bacillus subtilis.

[0112] The growth medium used for preparing the fermentate is any medium comprising necessary nutrients suitable for propagating the microorganism(s) suitable for use in the present invention. Suitable nutrients include but are not limited to amino peptides, peptides, yeast extract, salts, sugars, carbohydrates and / or vitamins. The medium can be based on dairy products, such as milk, cereals, fruits and / or vegetables.

[0113] In one aspect, the fermentates for use in the present invention includes fermentates from the microorganism Bacillus subtilis, wherein said B. subtilis is selected from a Bacillus subtilis strain having a 16S ribosomal RNA sequence displaying 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%sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of Bacillus subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof

[0114] Fermentates can be further concentrated prior to be processed into fermentate extracts or included in a composition to obtain an effective amount of actives in said fermentate. Fermentate extracts can be produced from the fermentates as described herein. Fermentates, fermentate extracts and fractions thereof can be spray-dried or lyophilized prior to be include in a skin care composition or skin care porduct.

[0115] It will be apparent that the fermentate may be used directly in the compositions and methods of the present invention, or that one or more fractions or extracts from said fermentate comprising actives may be isolated form the fermentate by any suitable means prior to use.

[0116] In one aspect of the invention, fermentate extracts are provided. Fermentate extracts for use in the present invention include fermentate extracts from the microorganism Bacillus subtilis.

[0117] As used herein, the term "fermentate extract” refers to an extract or fraction from a fermentate, wherein the fermentate was produced by propagating living microorganisms (microbial strains) in a nutrient medium as described above. In one aspect the fermentate extract is an extract of a fermentate produced by propagating of a Bacillus subtilis species.

[0118] In one aspect, the fermentate extract is a cell free supernatant of a fermentate. As used herein, a “cell free supernatant” 'fermentate supernatant”, “cell free fermentate” or “fermentate filtrate” are used interchangeably and refer to a fermentate extract that is substantially free of viable cells, such as a supernatant of a cell culture of at least one microorganism from which the cells have been removed. It is understood that cells can be removed from the cell culture by any method known in the art and that such removal of cells (such as through centrifugation, filtration) may still result in cell free supernatants that can comprise a trace amount of cells or cell debris. Methods for separating cells from growth media are well known in the art and can rely upon physical methods, for example, centrifugation to produce a cell pellet and a culture supernatant, filtration, ultrafiltration, tangential flow-filtration, normal flow filtration or reverse osmosis. Alternatively, or in addition, the separation method can be ligand-based and include, for example, an antibody that specifically binds to Bacillus subtilis. The antibody can be coupled to a solid support such as a magnetic bead. In one embodiment the cell free supernatant is obtained by filtration or centrifugation of the culture medium in which Bacillus subtilis cells were cultivated.

[0119] In one aspect, the cell free supernatant (also referred to as fermentate extract) is obtained by filtration or centrifugation of a Bacillus subtilis fermentate . In one aspect, cells were removed from the Bacillus subtilis fermentate by pelleting cells (centrifuge at 4,000 to 8,000 x g) and passing the supernatant through a 0.2 pM filter, to obtain an essentially cell-free supernatant.

[0120] In one aspect, the fermentate extract for use in the present invention is a Bacillus subtilis fermentate extract consisting essentially of cell free fermentate. The term "consisting essentially of" in the context of the fermentate includes that at least 90% of the fermentate have the indicated property (e.g. being cell free fermentate). Suitably at least 95% have the indicated property. Suitably at least 97% have the indicated property. Suitably at least 99% have the indicated property. In some embodiments at least 100% have the indicated property.

[0121] The fermentate extract for use in the compositions and methods and / or uses of the present invention may be substantially free of viable Bacillus subtilis cells, typically containing zero (or substantially zero) viable cells / mL fermentate.

[0122] In another aspect, the fermentate extract is an extract of a cell pellet obtained from a fermentate by pelleting the cells. Cells pellets can be obtained by centrifugation of the fermentate and removing the cell free supernatant.

[0123] In one aspect, the cell pellet extract is obtained from a Bacillus subtilis fermentate by pelleting the cells (centrifuge at 4,000 to 8,000 x g) and pouring off the supernatant, to leave behind the portion of cell pellet. The pellet is resuspended in acidic water (1 / 10 v / v) to a pH 2.0 - 4.0, vortexed, pelleted by centrifugation (4,000 to 8,000 x g) and the extract liquid is passed through a 0.2 pM filter. Optionally, the cell pellet can be resuspended in alkaline water (1 / 10 v / v) at a pH higher than 8.5. The cell pellet can be suspended in any solution or under any condition that produces an effective amount of the fermentate extract solution.

[0124] Starting from a cell pellet has the advantage that the volume of the resuspension liquid (extract volume) can be determined to produce an effective amount of the active extract and / or further concentrated to produce an effective amount of the active extract. Furthermore, resuspending the pellet in water or nonfermentation broth liquids, results in reducing or eliminating the non-active ingredients of the fermentation broth which may interfere with the actives of the cell pellet extract. In another aspect, Bacillus subtilis fermentate extracts are produced by combining the cell free supernatant fraction described above with the cell pellet extract described above.

[0125] In yet another aspect, the Bacillus subtilis fermentate extract is a whole broth fermentate extract that is prepared by first adjusting the pH of the fermentate (total fermentation broth) to a pH between 2.0 - 4.0 prior to pelleting out the insoluble cellular matter and optionally filtering supernatant through a 0.2 pM filter to yield a cell free supernatant referred to as “whole broth fermentate extract”.

[0126] Fermentate extracts can be further concentrated or purified prior to be included in a composition to obtain an effective amount of the fermentate extract. Fermentate extracts can be spray-dried or lyophilized prior to be include in a skin care composition or skin care product.

[0127] Alternatively, the Bacillus subtilis whole broth fermentate extract is a fermentate extract that was prepared by first adjusting the pH of the fermentate (total fermentation broth) to an alkaline pH prior to pelleting out the insoluble cellular matter and optionally filtering the supernatant through a 0.2 pM filter to yield a cell free supernatant (also referred to as “whole broth fermentate extract”).

[0128] In one aspect the fermentate extract is obtained from a fermentate that was produced with a nutrient medium having a pH between 2-12.

[0129] In one aspect the fermentate extract is obtained from a fermentate wherein the pH of the fermentate was adjusted to a pH between 2-12, prior to obtaining the fermentate extract.

[0130] It is also understood that production of a fermentate and fermentate extract can vary from batch to batch (fermentation to fermentation), which can result in different efficacies of the fermentate extract. As such batch to batch variation of the production of an effective amount of Bacillus subtilis fermentate extracts can be observed.

[0131] In one embodiment, the Bacillus subtilis fermentate, fermentate extract, or composition comprising the fermentate extract is formulated in a dry formulation or a liquid formulation. In one embodiment, the Bacillus subtilis fermentate, fermentate extract, or composition comprising the fermentate extract is formulated in at least one form selected from the group consisting of a loose or compact powder, a granule, a liquid suspension or solution, a spray solution, or any combination thereof.

[0132] In one aspect the granule comprises the Bacillus subtilis fermentate extract described herein at about 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 up to 100% by weight relative to a total weight of said granule.

[0133] Granules can be produced by any means known in the art, such as but not excluding, spray drying fermentate extract on a core or spray drying the fermentate to form a granule itself. In one aspect the granule is a readily dispersible layered granule. The layered granule can comprises a core surrounded by a coating layer that includes at least one effective amount of Bacillus subtilis fermentate extract distributed within a protectant matrix, and wherein the core is water-soluble and fast dissolving. The protectant matrix can include at least one polyhydroxy compound and at least one phosphate compound

[0134] In one aspect, the Bacillus subtilis fermentate, Bacillus subtilis fermentate extract, or fraction thereof, has efficacy in providing at least one skin care benefit selected from the group consisting of reducing a dandruff condition, reducing the occurrence 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 a skin or scalp disorder, skin barrier strengthening, improving skin barrier function, skin moisturizing, and any one combination thereof.

[0135] Skin barrier strengthening or improving skin barrier function means that the skin (such as, but not limiting to, its permeability barrier) is somehow “tighter / stronger” and it limits or avoids compounds from getting in (such as microbes, pollutants etc..) but also that it limits or avoids the amount of water that gets out, i.e. the skin barrier function gets improved.

[0136] In one aspect, the fermentate extracts, or fractions thereof, for use in the present invention includes fermentate extracts, or fractions thereof, from the microorganism Bacillus subtilis, wherein said Bacillus subtilis is selected from a Bacillus subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of Bacillus subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0137] As used herein, the term “fraction of Bacillus subtilis" or “fraction of Bacillus subtilis fermentate” or “fraction of Bacillus subtilis fermentate extract" or “fraction thereof” more particularly denotes a fragment of a Bacillus subtilis fermentate or Bacillus subtilis fermentate extract, which has efficacy in providing at least one skin care benefit selected from the group consisting of reducing a dandruff condition, reducing the occurrence 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 a skin or scalp disorder, skin barrier strengthening, improving skin barrier function, skin moisturizing, and any one combination thereof.

[0138] Skin care compositions and skin care products for providing at least one skin care benefit.

[0139] As used herein the term “skin care composition” refers to a composition comprising at least one skin care benefit agent capable of providing a skin care benefit.

[0140] As used herein the term “skin care benefit agent” or “active agent” or “bioactive” are used interchangeably, and refer to a Bacillus subtilis fermentate, Bacillus subtilis fermentate extract, or fraction thereof, that can provide a skin care (scalp care) benefit.

[0141] In some embodiments provided herein, the skin care benefit agent includes an effective amount of a Bacillus fermentate, Bacillus fermentate extract, or fraction thereof, wherein said Bacillus is selected from a Bacillus subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of Bacillus subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0142] As used herein the term “skin care benefit” refers to a benefit provided by a skin care benefit agent such as a fermentate, fermentate extract, or fraction thereof (or provided by a skin care composition and / or skin care product comprising an effective amount of said skin care benefit agent) when applied topically to a skin or sclap. Unexpectedly, the inventors have observed that a fermentate extract (cell free supernatant) of Bacillus subtilis (such as but not limiting to Bacillus subtilis N01 , Bacillus subtilis PC01 , Bacillus subtilis G01 , Bacillus subtilis G01_spo, and Bacillus subtilis K01 ) reduces the growth of Malassezia species. The inventors have also unexpectedly observed that a fermentate extract (cell free supernatant) of Bacillus subtilis (such as but not limiting to B. subtilis G01 and B. subtilis G01_spo) not only reduces the growth of Malassezia species but does so without affecting the growth of Staphylococcus hominis, Staphylococcus epidermis and Cutibacteria acnes strains. Furthermore, the inventors have unexpectedly observed that a fermentate extract (fermentate supernatant) of Bacillus subtilis (such as but not limitig to Bacillus subtilis G01 and Bacillus subtilis N01 ) increases Transepidermal Electrical Resistance (TEER) and increases expression of genes involved in fatty acid synthesis, cornification, small proline rich protein synthesis which is an indication of improved skin barrier function and strengthening of the skin barrier and therefore has suitable use in skin moisturization, strengthening of the skin barrier, improving skin barrier function, reducing dandruff, and reducing skin and scalp disorders.

[0143] In one aspect of the invention the skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturizing (protecting the skin against dehydration by maintaining, restoring and / or improving skin moisturization), promoting skin repair, and any one combination thereof.

[0144] For the purpose of the present disclosure, the term “dandruff” or “dandruff condition” refers to a condition manifested by a scalp presenting excessive dryness or excessive secretion of sebum, which, depending on the case, may be characterized by the presence of dry or greasy or oily dandruff, or even pruritis and / or an inflammation of the epidermis.

[0145] For the purpose of the present disclosure, the term “scalp or skin disorder” includes a dandruff condition of the scalp (seborrheic dermatitis), unbalanced microflora of the scalp, discomfort of the scalp, tinea versicolor, dry skin, irritated skin, or any one combination thereof. Dry dandruff conditions reflect a xerosis of the scalp, which may be combined with excessively rapid renewal of its stratum corneum. Dry dandruff flakes are generally in the form of small white or grey flakes and are spread over the scalp and on the clothing, giving rise to an unaesthetic visual effect.

[0146] The itching associated with dryness of the scalp may lead to erythema or even inflammation.

[0147] Greasy or oily dandruff conditions are one of the forms of seborrhoeic dermatitis. Individuals suffering from seborrhoeic dermatitis have an erythematous scalp covered with large, greasy or oily, yellow scales which accumulate so as to form packets. They have a pruritic scalp, and often have burning sensations on the affected areas. These phenomena may be amplified by the presence of pathogenic microorganisms, especially Malassezia species (Malassezia spp.). These microorganisms having the property of releasing fatty acids from the sebum may impair the barrier function of the epidermis and give rise to inflammation.

[0148] During dandruff conditions of the scalp, the cutaneous barrier is unbalanced, its integrity and its hydration are impaired, and its microflora is disturbed. The skin of the scalp is irritated and pruritic, brittle, less hydrated, and sensitive to infections.

[0149] Malassezia species described herein include, but are not limited to, Malassezia restricta (M. restricta), Malassezia globosa (M. globosa), Malassezia furfur (M. furfur), Malassezia sympodialis (M. sympodialis), Malassezia phylotype 5 (M. phylotype 5), other uncharacterized Malassezia species, and any combination thereof.

[0150] Human skin is colonized by a diverse microbiota (Byrd, A., Belkaid, Y. & Segre, J. The human skin microbiome. Nat Rev Microbiol 16, 143-155; 2018). . Staphylococcus hominis, Staphylococcus epidermis and Cutibacteria acnes strains (formerly known as Propioni bacterium) are two of the dominant commensal species.

[0151] As used herein, the term “biofilm” refers to a community of microorganisms embedded in an extracellular polymer matrix attached to a surface. The extracellular polymer matrix is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. A biofilm may have one or more microorganisms and further includes water and may include other trapped particles. The microorganisms may be gram positive or gram-negative bacteria (aerobic or anaerobic); algae, protozoa, and / or yeast or filamentous fungi. In one embodiment, the biofilm is living cells including one or more Malassezia species.

[0152] As used herein, “surface” means any structure having sufficient mass to allow for attachment of biofilm. A surface includes a hard surface and a soft surface. Hard surfaces include, but are not limited to metal, glass, ceramics, wood, minerals (rock, stone, marble, granite), aggregate materials such as concrete, plastics, composite materials, hard rubber materials, and gypsum. Other surfaces may be biological surfaces, such as skin, scalp, or keratin.

[0153] Additional benefit agents for skin care can include antidandruff active agents.

[0154] Examples of antidandruff active agents include keratolytic agents such as salicylic acid and sulphur in its various forms, regulators of keratinization such as zinc pyrithione, a pyridinethione salt, a trihalocarbamide, triclosan, an azole compound, an antifungal polymer, allantoin, steroids such as topical corticosteroids, tar or polytar (coal tar), undecylenic acid, fumaric acid, an allylamine and mixtures thereof, ciclopirox, octopirox, piroctone olamine, clobetasol propionate, betamethasone valerate, tea tree oil, a mixed oil of thyme and catnip, topical antifungals such as selenium sulfide, imidazole (e.g. ketoconazole), hydroxypyridones (e.g. ciclopirox), naturopathic agents such as Melaleuca sp. oil, Aloe vera, and probiotic microorganisms. (Indian J. Dermatol, 2010 Apr-Jun; 55(2): 130-134).

[0155] In one aspect, the skin care benefit agents are selected from the group consisting of a cell free supernatant, a cell pellet extract, and / or a whole broth fermentate extract obtained from a fermentate of a Bacillus subtilis selected from the group consisting of a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of 8. subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0156] The skin care benefit agents described herein, or any effective amount of said skin care benefit agents described herein, can be formulated in a skin care composition.

[0157] In one aspect, the skin care benefit agent consisting of an effective amount of a Bacillus subtilis fermentate, Bacillus subtilis fermentate extract, and / or a fraction thereof, is formulated in a skin care composition. 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0158] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein said skin barrier strengthening comprises administering the composition to the subject’s skin or scalp to strengthen the skin barrier of said subject.

[0159] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein said improving skin barrier function comprises administering the composition to the subject’s skin or scalp to improve the skin barrier function of said subject.

[0160] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein said skin moisturization comprises administering the composition to the subject’s skin or scalp to moisturize the skin or scalp of said subject.

[0161] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein said composition comprises an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, at 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%,

[0162] 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%,

[0163] 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %,

[0164] 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%,

[0165] 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,

[0166] 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%,

[0167] 94%, 95%, 96%, 97%, 98%, 99% up to 100 % by weight relative to a total weight of said composition.

[0168] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein said composition comprises an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, at 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%,

[0169] 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,

[0170] 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%,

[0171] 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%,

[0172] 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %,

[0173] 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% up to 100 % by volume relative to a total volume of said composition.

[0174] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein the Bacillus subtilis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus subtilis fermentate, a cell pellet extract obtained from a Bacillus subtilis fermentate, a whole broth fermentate extract obtained from a Bacillus subtilis fermentate, and any one combination thereof.

[0175] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein the scalp or skin disorder is selected from the group consisting of an unbalanced ecoflora of the scalp, discomfort of the scalp, tinea versicolor, dry skin, irritated skin, and any one combination thereof.

[0176] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein said reducing of dandruff comprises administering the composition to the subject’s skin or scalp to reduce dandruff.

[0177] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein the Bacillus subtilis fermentate extract, or fraction thereof, is obtained from a fermentate of a Bacillus subtilis selected from the group consisting of a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0178] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, further comprising one or more antidandruff active agents.

[0179] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein the composition reduces growth of Malassezia species without affecting growth of Staphylococcus hominis, Staphylococcus epidermis and Cutibacte a acnes strains.

[0180] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein said skin barrier strengthening and / or said improving skin barrier function and / or said skin moisturization occurs through fatty acid synthesis, cornification, small proline rich proteins synthesis, and any one combination thereof.

[0181] In one aspect, the fatty acid synthesis or increased fatty acid synthesis (when compared to the same skin care composition lacking said effective amount of a Bacillus subtilis fermentate extract, or fraction thereof) occurs through elongation of fatty acids.

[0182] In one aspect, the fatty acid synthesis or increased fatty acid synthesis (when compared to the same skin care composition lacking said effective amount of a Bacillus subtilis fermentate extract, or fraction thereof) occurs through elongation of fatty acids, wherein said elongation of fatty acids occurs through increased expression (when compared to the same skin care composition lacking said effective amount of a Bacillus subtilis fermentate extract, or fraction thereof) of Elongation of Very Long Chain Fatty Acid (ELOVL) fatty acid elongase 7 (ELVOL7).

[0183] In one aspect, the cornification or increased cornification (when compared to the same skin care composition lacking said effective amount of a Bacillus subtilis fermentate extract, or fraction thereof) occurs through an increased expression (when compared to the same skin care composition lacking said effective amount of a Bacillus subtilis fermentate extract, or fraction thereof) of a late cornified envelope 1 c (LCE1 C) protein.

[0184] In one aspect, the small proline rich proteins synthesis or increased small proline rich proteins synthesis (when compared to the same skin care composition lacking said effective amount of a Bacillus subtilis fermentate extract, or fraction thereof) occurs through increased expression of small proline rich proteins (SPRR), such as but not limiting to, SPRRs selected from the group consisting of SPRR2F, SPRR2E, SPRR2A, SPRR2D, and any one combination thereof.

[0185] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises at least one active agent of less than 3 Kda.

[0186] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises between 0.1 % to 100% of at least one active agent of less than 3 Kda on a weight basis relative to a total weight of said effective amount.

[0187] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises between 0.1 % to 100% of at least one active agent of less than 3 Kda on a volume basis relative to a total volume of said effective amount. In one embodiment, the skin care composition is a skin care composition as dexcribed herein, wherein said at least one active agent is bacilysin.

[0188] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, growth inhibition of Malassezia, growth of Malassezia species without affecting growth of Staphylococcus hominis, Staphylococcus epidermis and Cutibacteria acnes strains and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises at least one active agent of less than 3 Kda.

[0189] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, growth inhibition of Malassezia, growth of Malassezia species without affecting growth of Staphylococcus hominis and Cutibacteria acnes strains and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises between 0.1 % to 100% of at least one active agent of less than 3 Kda on a volume basis relative to a total volume of said effective amount.

[0190] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, growth inhibition of Malassezia, growth of Malassezia species without affecting growth of Staphylococcus hominis and Cutibacteria acnes strains and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises between 0.1 % to 100% of at least one active agent of less than 3 Kda on a weight basis relative to a total weight of said effective amount.

[0191] In one embodiment, the skin care composition is a skin care composition described herein, wherein said at least one active agent is bacilysin.

[0192] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate 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 one combination thereof.

[0193] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises an active agent of less than 3 Kda.

[0194] It will be further understood that the skin care composition for use in the present invention may further comprise one or more of probiotic bacteria in addition to the active agents from Bacillus subtilis (such as the Bacillus subtilis fermentate, Bacillus subtilis fermentate extract, and / or the Bacillus subtilis fermentate fraction).

[0195] The skin care composition can comprise additional compounds selected from the group consisting of consisting of a fragrance, an excipient, a preservative, a pH adjuster, an aqueous carrier, an alcohol carrier, an emollient, a solidification agent, a hydration agent, an emulsifier, a solubilizer, a surfactant, a thickening agent, a salt and any one combination thereof.

[0196] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, the composition further comprising an additional compound selected from the group consisting of a fragrance, an excipient, a preservative, a pH adjuster, an aqueous carrier, an alcohol carrier, an emollient, a solidification agent, a hydration agent, an emulsifier, a solubilizer, a surfactant, a thickening agent, a salt and any one combination thereof.

[0197] Preservatives include but are not limited to parabens, sodium benzoate, potassium sorbate, phenyl ethyl alcohol, Lauryl ethyl arginate (LAE) and any combination thereof. pH adjusters include but are not limited to weak acids, strong acids, any compound that can adjust the pH, such as but not limiting to citric acid, or any combination thereof.

[0198] In one embodiment, the composition is a Bacillus subtilis strain variant fermentate, Bacillus subtilis strain variant fermentate extract, or fraction of said variant fermentate, of a B. subtilis strain variant, wherein said Bacillus subtilis strain variant is derived from a parental B. subtilis by knocking out at least one sporulation gene of said parental B. subtilis strain, wherein said variant fermentate, said variant fermentate extract, or said fraction of said variant fermentate, has increased activity when compared to the activity of a fermentate, a fermentate extract, or fraction thereof derived from said parental B. subtilis strain, wherein said activity is selected from the group consisting of reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof. In one embodiment, the composition is a Bacillus subtilis strain variant fermentate, Bacillus subtilis strain variant fermentate extract, or fraction of said variant fermentate, of a B. subtilis strain variant, wherein said Bacillus subtilis strain variant is derived from a parental B. subtilis by knocking out a SpollE sporulation gene of said parental B. subtilis strain, wherein said variant fermentate, said variant fermentate extract, or said fraction of said variant fermentate, has increased activity when compared to the activity of a fermentate, a fermentate extract, or fraction thereof derived from said parental B. subtilis strain, wherein said activity is selected from the group consisting of reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0199] The skin care compositions described herein can be used in formulations and skin care products.

[0200] As used herein “skin care products” refer to products comprising the skin care compositions described herein, including but not limiting to cosmetic products, aqueous solutions, emulsions, serums, jellies, patches, lotions, topical moisturizers, creams, pastes, balms, ointments, pomades, gels, liquids, sprays, shampoos, foam, kits, or any one combinations thereof.

[0201] In one embodiment, the skin care product is a skin care product comprising the skin care composition described herein.

[0202] In one embodiment, the skin care product is a skin care product comprising the skin care composition described herein and one or more dermatologically or skin care acceptable component.

[0203] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof. In one embodiment, the skin care product is a product comprising a skin care composition for use in reducing a skin or scalp disorder, the composition comprising an effective amount of a Bacillus subtilis fermentate, Bacillus subtllis fermentate extract, and / or fraction thereof, wherein said composition reduces and / or treats said skin or scalp disorder, and one or more dermatologically or skin care acceptable component.

[0204] 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 thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the Bacillus subtilis is selected from the group consisting of a Bacillus subtilis having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO:1 ) of Bacillus subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a Bacillus subtilis having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO:2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a Bacillus subtilis suitable for use in the present invention also includes Bacillus subtilis having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO:3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a Bacillus subtilis having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO:7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0205] 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% up to 10% of the skin care composition described herein on a weight basis relative to a total weight of said skin care product.

[0206] 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% up to 10% of a skin care composition described herein on a volume basis relative to a total volume of said skin care product.

[0207] In one embodiment, the skin care product described herein is formulated for topical administration.

[0208] In one embodiment, the skin care product described herein is formulated for topical administration to the skin or scalp.

[0209] In one embodiment, the skin care product is a product comprising a skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein the skin care product is selected from the group consisting of a lotion, a serum, a jelly, a cream, a gel, a hydrogel, an emulsion, a solid cosmetic, a mask, a patch, a shampoo, a loose or compact powder, a liquid suspension or solution, a spray solution and a stick, or any combination thereof.

[0210] In one embodiment, the skin care product is selected from the group consisting of a lotion, a serum, a jelly, a cream, a gel, a hydrogel, an emulsion, a solid cosmetic, a mask, a patch, a shampoo, a loose or compact powder, a liquid suspension or solution, a spray solution and a stick.

[0211] In one embodiment, the skin care product is a lotion, a serum, a jelly, a cream, a gel, a hydrogel, an emulsion, a solid cosmetic, a mask, a patch, a shampoo, a loose or compact powder, a liquid suspension or solution, a spray solution or a stick 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% up to 10% of a skin care composition described herein on a weight basis relative to a total weight of said skin care formulation.

[0212] 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 a Bacillus subtilis fermentate, and / or fraction thereof wherein the second skin care composition comprises at least an effective amount of at least one second active agent selected from antidandruff active agents, for topical administration. Examples of such antidandruff active agents include keratolytic agents such as salicylic acid and sulphur in its various forms, regulators of keratinization such as zinc pyrithione, a pyridinethione salt, a trihalocarbamide, triclosan, an azole compound, an antifungal polymer, allantoin, steroids such as topical corticosteroids, tar or polytar (coal tar), undecylenic acid, fumaric acid, an allylamine and mixtures thereof, ciclopirox, octopirox, piroctone olamine, clobetasol propionate, betamethasone valerate, tea tree oil, a mixed oil of thyme and catnip, topical antifungals such as selenium sulfide, imidazole (e.g. ketoconazole), hydroxypyridones (e.g. ciclopirox), naturopathic agents such as Melaleuca sp. oil, Aloe vera, and probiotic microorganisms. (Indian J. Dermatol, 2010 Apr-Jun; 55(2): 130-134).

[0213] 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 an effective amount of a Bacillus subtilis fermentate, and / or fraction thereof; wherein the second skin care composition comprises at least an effective amount of at least one second active agent selected from antidandruff active agents, for topical administration, wherein the first skin care composition is formulated in at least one form selected from the group consisting of a gel, an emulsion, a hydrogel, a loose or compact powder, a liquid suspension or solution, or a spray solution.

[0214] The topical formulation for use in the present invention may be in any form suitable for application to the scalp or skin surface, such as a cream, lotion, sprays, solution, gel, ointment, paste, plaster, paint, bioadhesive, suspensions or the like, and / or may be prepared so as to contain liposomes, micelles, and / or microspheres. Such a formulation may be used in combination with an occlusive overlayer so that moisture evaporating from the body surface is maintained within the formulation upon application to the body surface and thereafter.

[0215] Topical formulations include those in which the active ingredient(s) is (are) dissolved or dispersed in a dermatological vehicle known in the art (e.g. aqueous or non-aqueous gels, ointments, water-in-oil or oil-in-water emulsions). Constituents of such vehicles may comprise water, aqueous buffer solutions, nonaqueous solvents (such as ethanol, isopropanol, benzyl alcohol, 2-(2- ethoxyethoxy) ethanol, propylene glycol, propylene glycol monolaurate, glycofurol or glycerol), oils (e.g. a mineral oil such as a liquid paraffin, natural or synthetic triglycerides, or silicone oils such as dimethicone). Depending, inter alia, upon the nature of the formulation as well as its intended use and site of application, the dermatological vehicle employed may contain one or more components (for example, when the formulation is an aqueous gel, components in addition to water) selected from the following list: a solubilizing agent or solvent (e.g. a [3- cyclodextrin, such as hydroxypropyl [3-cyclodextrin, or an alcohol or polyol such as ethanol, propylene glycol or glycerol);a thickening agent (e.g. hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose or carbomer);a gelling agent (e.g. a polyoxyethylene-polyoxypropylene copolymer);a preservative (e.g. benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorbutol, a benzoate, potassium sorbate or EDTA or salt thereof); and pH buffering agent(s) (such as a mixture of dihydrogen phosphate and hydrogen phosphate salts, or a mixture of citric acid and a hydrogen phosphate salt).

[0216] A skin care product includes a liquid lotion (true solution) comprising water as a solvent and water-soluble additives (solutes), such as but not limiting to an active, a fragrance, a color, a preservative, a pH adjuster, a chelating agent, or any one combination thereof.

[0217] A skin care product includes a dispersion such as an emulsion (such as, but not limited to the following: liquid in liquid [water in oil W / O, O / W, W / O / W], suspension [solid / liquid or liquid / solid], aerosol [liquid / gas or solid / gas], foam / mousse [gas / liquid or gas / emulsion, or gas / solid]). An example of an Oil in Water [O / W] emulsion includes, but is not limited to a combination of a water phase, an emulsifier, a fatty phase and an at least one additive. The water phase can comprise water, humectants and stabilizing agents [such as, but not limiting to, synthetic polymers, carbomers, natural polymers, xanthan gum, acacia gum, carragheenan, gellan, or any one combination thereof). Emulsifiers include, but are not limited to, anionic emulsifiers, cationic emulsifiers, non-ionic emulsifiers, amphoteric emulsifiers, silicone emulsifiers), auto emulsifying agents. Fatty phases (lipophilic ingredients) include, but are not limited to, waxes, butter, fatty esters, triglycerides, vegetal oil, mineral oil (parffinum), silicones, and thickeners / oil jellifying agents. Additives include, but are not limited to, preservative, fragrance (most often lipophilic), color, anti-oxidant, chelating agent, actives, pH adjuster (citric acid, lactic acid, AHA), neutralizers / strong basic agent like NaOH, Trimethylamine (for acrylic polymers to jellify) and powders.

[0218] A skin care product includes an aqueous gel comprising a water phase (including water, humectants, actives), a jellifying agent (such as but not limited to synthetic polymers, natural polymers, xanthan gum, acacia gum, carragheenan, gellan) and an additive (such as but not limited to fragrance, high HLB surfactant, color, actives, preservative system, pH adjuster, neutralizing agent, powders).

[0219] A skin care product includes a cleansing I surfactant system (such as but not limited to a shampoo, shower gel, micellar water) comprising a water phase (water, humectants), a surfactant, an additive (such as but not limited to fragrance, high HLB surfactant, color, actives, preservative system, pH adjuster, neutralizing agent, powders) and optionally a jellifying agent (such as but not limited to synthetic polymers, natural polymers, xanthan gum, acacia gum, carragheenan, gellan).

[0220] A dermatologically or skin care acceptable carrier may also be incorporated in the skin care product (formulation) of the present invention and may be any carrier conventionally used in the art. Examples thereof include water, lower alcohols, higher alcohols, polyhydric alcohols, monosaccharides, disaccharides, polysaccharides, hydrocarbon oils, fats and oils, waxes, fatty acids, silicone oils, nonionic surfactants, ionic surfactants, silicone surfactants, and water-based mixtures and emulsion-based mixtures of such carriers.

[0221] The term " dermatologically acceptable" or " dermatologically acceptable carrier" or “skin care acceptable” or “skin care acceptable carrier” is used herein to refer to a compound or composition that may be incorporated into a dermatologically or skin care formulation without causing undesirable biological effects or unwanted interaction with other components of the formulation.

[0222] "Carriers" or "vehicles" as used herein refer to carrier materials suitable for incorporation in a topically applied composition. Carriers and vehicles useful herein include any such materials known in the art, which are nontoxic and do not interact with other components of the formulation in which it is contained in a deleterious manner.

[0223] The term "aqueous" refers to a formulation that contains water or that becomes water-containing following application to the skin or mucosal tissue.

[0224] Skin care products described herein may further comprise one or more dermatologically or skin care acceptable components known or otherwise effective for use skin care, provided that the optional components are physically and chemically compatible with the essential components described herein, or do not otherwise unduly impair product stability, aesthetics, or performance. Non-limiting examples of such optional components are disclosed in International Skin Care Ingredient Dictionary, Ninth Edition, 2002, and CTFA Skin Care Ingredient Handbook, Tenth Edition, 2004.

[0225] In one aspect, the dermatologically or skin care acceptable component is a dermatologically acceptable carrier comprising from about 10 wt.% to about 99.9 wt.%, alternatively from about 50 wt.% to about 95 wt.%, and alternatively from about 75 wt.% to about 95 wt.%, of a dermatologically acceptable carrier. Carriers suitable for use with the composition(s) may include, for example, those used in the formulation of mousses, tonics, gels, skin moisturizers and lotions. The carrier may comprise water; organic oils; silicones such as volatile silicones, amino or non-amino silicone gums or oils, and mixtures thereof; mineral oils; plant oils such as olive oil, castor oil, rapeseed oil, coconut oil, wheat germ oil, sweet almond oil, avocado oil, macadamia oil, apricot oil, safflower oil, candlenut oil, false flax oil, tamanu oil, lemon oil and mixtures thereof; waxes; and organic compounds such as C2-C10 alkanes, acetone, methyl ethyl ketone, volatile organic C1-C12 alcohols, esters of C1-C20 acids and of Ci-Cs alcohols such as methyl acetate, butyl acetate, ethyl acetate, and isopropyl myristate, dimethoxyethane, diethoxyethane, C10-C30 fatty alcohols such as lauryl alcohol, cetyl alcohol, stearyl alcohol, and behenyl alcohol; C10-C30 fatty acids such as lauric acid and stearic acid; C10-C30 fatty amides such as lauric diethanolamide; C10-C30 fatty alkyl esters such as C10-C30 fatty alkyl benzoates; hydroxypropylcellulose, 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 of ordinary skill in the art.

[0226] The skin care products described herein may further comprise 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 the composition(s). 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 trideceth-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 ethoxylate urethane polymers (e.g., UCARE™ Polyphobe Series of alkali swellable polymers commercially available from Union Carbide); and combinations thereof. In this context, the term “unneutralized” means that the optional polymer and copolymer gelling agent materials contain unneutralized acid monomers.

[0227] The dermatologically or skin care acceptable medium may contain a fatty substance in a proportion generally of from about 10 to about 90% by weight relative to the total weight of the product, where the fatty phase containing at least one liquid, solid or semi-solid fatty substance. The fatty substance includes, but is not limited to, oils, waxes, gums, and so-called pasty fatty substances. Alternatively, the products may be in the form of a stable dispersion such as a water-in-oil or oil-in-water emulsion. Additionally, the skin care products may contain one or more conventional skin care or dermatological additives or adjuvants, including but not limited to, antioxidants, preserving agents, fillers, surfactants, UVA and / or UVB sunscreens, fragrances, thickeners, wetting agents and anionic, nonionic or amphoteric polymers, and dyes or pigments (colorant agents).

[0228] The dermatologically acceptable carrier may be a moisturizer formulation containing at least one emulsifiers, at least one surfactant, or any combination thereof.

[0229] Skin care compositions and skin care products can further comprise skin care active ingredient materials including sun screen agents, moisturizers, humectants, benefiting agents skin, depositing agents such as surfactants, occlusive agents, moisture barriers, lubricants, emollients, anti-aging agents, antistatic agents, abrasive, antimicrobials, conditioners, exfoliants, fragrances, viscosifying agents, salts, lipids, phospholipids, vitamins, foam stabilizers, pH modifiers, preservatives, suspending agents, silicone oils, silicone derivatives, essential oils, oils, fats, fatty acids, fatty acid esters, fatty alcohols, waxes, polyols, hydrocarbons, and mixtures thereof.

[0230] Other ingredients that may be included in a skin care composition or skin care product include, without limitation, at least one active ingredient for the reduction or prevention of skin ailments, providing a skin care effect, or for providing a moisturizing benefit to skin, such as zinc oxide, petrolatum, white petrolatum, mineral oil, cod liver oil, lanolin, dimethicone, hard fat, vitamin A, allantoin, calamine, kaolin, glycerin, or colloidal oatmeal, and combinations of these, one or more natural moisturizing factors (such as ceramides, hyaluronic acid, glycerin, squalane, amino acids, cholesterol, fatty acids, triglycerides, phospholipids, glycosphingolipids, urea, linoleic acid, glycosaminoglycans, mucopolysaccharide, sodium lactate, or sodium pyrrolidone carboxylate, for example), glycerides, apricot kernel oil, canola 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.

[0231] Any number of dermatologically acceptable materials commonly used in skin care products may also be incorporated into the present skin care products such as skin conditioning agents and skin colorants.

[0232] Skin conditioning agents as herein defined include, but are not limited to astringents, which tighten skin; exfoliants, which remove dead skin cells; emollients, which help maintain a smooth, soft, pliable appearance; humectants, which increase the water content of the top layer of skin; occlusives, which retard evaporation of water from the skin’s surface; and miscellaneous compounds that enhance the appearance of dry or damaged skin or reduce flaking and restore suppleness. Skin conditioning agents are well known in the art, see for example Green et al. (W001 / 07009), and are available commercially from various sources. Suitable examples of skin conditioning agents include, but are not limited to, lactobionic acid, gluconic acid, alpha-hydroxy acids, beta-hydroxy acids, polyols, hyaluronic acid, D,L-panthenol, polysalicylates, vitamin A palmitate, vitamin E acetate, glycerin, sorbitol, silicones, silicone derivatives, lanolin, natural oils, xylitol, fucose, rhamnose, xylitol, betaine, and triglyceride esters. The skin conditioning agents may include polysalicylates, propylene glycol (CAS No. 57-55-6, Dow Chemical, Midland, Ml), glycerin (CAS No. 56-81 -5, Proctor & Gamble Co., Cincinnati, OH), glycolic acid (CAS No. 79-14-1 , DuPont Co., Wilmington, DE), lactic acid (CAS No. 50-21 -5, Alfa Aesar, Ward Hill, MA), malic acid (CAS No. 617- 48-1 , Alfa Aesar), citric acid (CAS No. 77-92-9, Alfa Aesar), tartaric acid (CAS NO. 133-37-9, Alfa Aesar), glucaric acid (CAS No. 87-73-0), galactaric acid (CAS No. 526-99-8), 3-hydroxyvaleric acid (CAS No. 10237-77-1 ), salicylic acid (CAS No. 69-72-7, Alfa Aesar), and 1 ,3 propanediol (CAS No. 504-63-2, DuPont Co., Wilmington, DE). Polysalicylates may be prepared by the method described by White et al. in U.S. Patent No. 4,855,483, incorporated herein by reference. Glucaric acid may be synthesized using the method described by Merbouh et al. (Carbohydr. Res. 336:75-78 (2001 ). The 3-hydroxyvaleric acid may be prepared as described by Bramucci in published international patent application number WO 02 / 012530.

[0233] Skin care compositions and skin care products can comprise skin care additives such as, but not limiting to, colorants / dyes, fragrances, actives, preservatives, pH adjusters, chelators, and antioxidants.

[0234] Skin care compositions and skin care products described herein can also be part of a kit for providing one or more skin care benefits such as, but not limiting to, a kit for reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization or any one combination thereof.

[0235] In one aspect the kit is a kit comprising a skin care product for the reduction of a dandruff condition of a subject in need, wherein said skin care product comprises an effective amount of a Bacillus subtilis fermentate extract described herein, and / or fraction thereof, and written instructions for administration said skin care product to the subject in need. Methods for providing at least one skin care benefit to a skin or scalp

[0236] The skin care compositions and skin care products described herein can be used in methods for providing at least one skin care benefit to a subject in need thereof.

[0237] In one aspect the skin care compositions and skin care products described herein can be used in methods for providing at least one skin care benefit to a subject in need thereof, wherein the skin care benefit is selected from the group consisting of reducing dandruff, reducing the occurrence 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 a skin or scalp disorder, skin barrier strengthening, improving skin barrier function, skin moisturizing (protecting the skin against dehydration by maintaining, restoring and / or strengthening the moisturization of the skin), and any one combination thereof.

[0238] In one embodiment, the method is a method for providing at least one skin care benefit to a subject in need thereof, the method comprising topically administering a skin care composition or skin care product comprising said composition to the skin or scalp of said subject, wherein said composition comprises an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing the occurrence 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 on the skin (scalp), reducing a skin or scalp disorder, skin barrier strengthening, improving skin barrier function, skin moisturizing (protecting the skin against dehydration by maintaining, restoring and / or strengthening the moisturization of the skin), and any one combination thereof. In one aspect, the Bacillus subtilis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus subtilis fermentate, a cell pellet extract obtained from a Bacillus subtilis fermentate, a whole broth fermentate extract obtained from a Bacillus subtilis fermentate, and any one combination thereof. In one embodiment, the method is a method for reducing dandruff in a subject, the method comprising topically administering a skin care composition or skin care product comprising said skin care composition to the scalp of said subject, wherein the skin care composition comprises an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof.

[0239] In one embodiment, the method is a method for reducing a scalp or skin disorder in a subject, the method comprising topically administering a skin care composition or skin care product comprising said skin care composition to the scalp of said subject, wherein the skin care composition comprises an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof. In one aspect, the scalp or skin disorder is selected from the group consisting of a dandruff condition of the scalp (seborrheic dermatitis), unbalanced ecoflora of the scalp, discomfort of the scalp, tinea versicolor, dry skin, irritated skin, and any one combination thereof.

[0240] In one embodiment, the method is a method for skin barrier strengthening in a subject, the method comprising topically administering a skin care composition or skin care product described herein, to the skin or scalp of said subject.

[0241] In one embodiment, the method is a method for skin moisturizing in a subject, the method comprising topically administering a skin care composition or skin care product described herein, to the skin or scalp of said subject.

[0242] In one aspect the Bacillus subtilis fermentate, Bacillus subtilis fermentate extract, or fraction thereof for use in the methods described herein, are obtained from a fermentate of a Bacillus subtilis selected from the group consisting of a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of 8. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0243] It will be further apparent that the skin care composition for use according to the present invention may comprise, 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%,

[0244] 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%,

[0245] 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%,

[0246] 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%,

[0247] 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 fermentate, Bacillus subtilis fermentate extract, and / or fraction thereof, on a volume basis relative to a total volume of said skin care composition.

[0248] In one aspect the skin care composition for use according to the present invention may comprise, 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%,

[0249] 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%,

[0250] 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%,

[0251] 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%,

[0252] 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%,

[0253] 99% up to 100 % of Bacillus subtilis fermentate, Bacillus subtilis fermentate extract, and / or fraction thereof, on a weight basis relative to a total weight of said skin care composition.

[0254] In one aspect, the skin care product is administered to a subject in need thereof, 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%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%,

[0255] 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%,

[0256] 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% up to 50% of a skin care composition described herein on a weight basis relative to a total weight of said skin care product

[0257] In one aspect, the skin care composition or skin care product for use according to the present invention is selected from the group consisting of a lotion, a serum, a jelly, a cream, a gel, an emulsion, a solid cosmetic, a mask, a patch, a shampoo, and a stick Methods for increasing the activity of a Bacillus subtilis strain fermentate extract and compositions thereof

[0258] Methods for increasing the activity of a Bacillus subtilis strain fermentate extract for providing at least one skin care benefit are disclosed herein.

[0259] The inventors have unexpectedly observed that when one knocks out a sporulation gene, such as but not limiting to SpollE, in a parental B. subtilis strain (such as but not limiting to B. subtilis E04) to produce a B. subtilis variant strain (such as but not limiting to B. subtilis E04_spo), an increased activity of the fermentate, fermentate extract, or fraction thereof derived from said B. subtilis variant strain was observed when compared to the activity of the fermentate, fermentate extract, or fraction thereof, derived from the parental B. subtilis strain, wherein said activity is selected from the group consisting of reducing dandruff, reducing the occurrence of Malassezia species species on the skin or scalp, skin barrier strengthening, improving skin barrier function, and any one combination thereof.

[0260] In one embodiment, the method a method for increasing the activity of a Bacillus subtilis strain fermentate 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 fermentate extract for providing at least one skin care benefit selected from the group consisting of reducing dandruff, reducing the occurrence of Malassezia species species on the skin or scalp, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof;(b) modifying said parent Bacillus subtilis strain by knocking out at least one sporulation gene from the genome of said Bacillus subtilis strain, thereby producing a variant strain that is unable to form spores; and, (c) producing a fermentate extract of said Bacillus subtilis variant strain, wherein said fermentate extract of said Bacillus subtilis variant strain has an increased activity for providing at least one skin care benefit selected from the group consisting of reducing dandruff, reducing the occurrence of Malassezia species species on the skin or scalp, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof, when compared to the activity of the fermentate extract of said parent Bacillus subtilis strain.

[0261] In one embodiment, the method a method for increasing the activity of a Bacillus subtilis strain fermentate 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 fermentate extract for providing at least one skin care benefit selected from the group consisting of reducing dandruff, reducing the occurrence of Malassezia species species on the skin or scalp, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof;(b) modifying said parent Bacillus subtilis strain by knocking out a SpollE sporulation gene from the genome of said Bacillus subtilis strain, thereby producing a variant strain that is unable to form spores; and, (c) producing a fermentate extract of said Bacillus subtilis variant strain, wherein said fermentate extract of said Bacillus subtilis variant strain has an increased activity for providing at least one skin care benefit selected from the group consisting of reducing dandruff, reducing the occurrence of Malassezia species species on the skin or scalp, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof, when compared to the activity of the fermentate extract of said parent Bacillus subtilis strain.

[0262] General definitions

[0263] The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety.

[0264] In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise.

[0265] As used herein, the articles “a”, “an”, and “the” preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances ( / .e., occurrences) of the element or component. Therefore “a”, “an”, and “the” should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular. When an amount, concentration, or other value or parameter is given either as a range, preferred range, or a list of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope be limited to the specific values recited when defining a range.

[0266] The use of numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both proceeded by the word “about”. In this manner, slight variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. Also, the disclosure of these ranges is intended as a continuous range including each and every value between the minimum and maximum values. As used herein, the term “about” modifying the quantity of an ingredient or reactant employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term “about” also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term “about”, the claims include equivalents to the quantities.

[0267] As used herein “administer” or “administering” is meant the action of introducing one or more microorganism (microbial strain), fermentates, fermentate extracts or fractions thereof, skin care composition(s), skin care formulation(s) and / or skin care product(s) to a subject in need for providing at least one skin care benefit, such as but not limiting to, reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0268] Administering one or more microorganism (microbial strain), fermentates, fermentate extracts or fractions thereof, skin care composition(s), skin care formulation(s) and / or skin care product(s) to a subject includes applying or introducing one or more microorganism (microbial strain), fermentates, fermentate extracts or fractions thereof, skin care composition(s), skin care formulation(s) and / or skin care product(s) to a scalp, a skin surface, and to in-vitro or in-vivo skin cells.

[0269] As used herein, the term “biological contaminants” refers to one or more unwanted and / or pathogenic biological entities including, but not limited to, microorganisms, spores, viruses, prions, and mixtures thereof.

[0270] As used herein, the term “comprising” means the presence of the stated features, integers, steps, or components as referred to in the claims, but that it does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof. The term “comprising” is intended to include embodiments encompassed by the terms “consisting essentially of” and “consisting of”. Similarly, the term “consisting essentially of’ is intended to include embodiments encompassed by the term “consisting of”.

[0271] As used herein, the term “embodiment” or “disclosure” is not meant to be limiting, but applies generally to any of the embodiments defined in the claims or described herein. These terms are used interchangeably herein.

[0272] As used herein, the term “excipient” refers to inactive substance used as a carrier for active ingredients, in a formulation. The excipient may be used to stabilize the active ingredient in a formulation, such as the storage stability of the active ingredient. Excipients are also sometimes used to bulk up formulations that contain active ingredients. An “active ingredient” includes a skin care benefit agent as described herein.

[0273] As used herein, the term “effective amount” refers to the amount sufficient to obtain the desired effect. A desired effect includes, but is not limited to, reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0274] As used herein, “prevent,” “preventing,” “prevention” and grammatical variations thereof refers to a method of partially or completely delaying or precluding the onset or recurrence of a disorder or condition (such as a scalp disorder, dry skin, skin irritation) and / or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a disorder or condition or reducing a subject’s risk of acquiring or reacquiring a disorder or condition or one or more of its attendant symptoms.

[0275] As used herein, the term “reducing”, “reduces” and grammatical variations thereof in relation to a particular trait, characteristic, feature, biological process, or phenomena refers to a decrease in the particular trait, characteristic, feature, biological process, or phenomena. The trait, characteristic, feature, biological process, or phenomena can be decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater than 100%.

[0276] The terms “percent by weight”, “weight percentage (wt.%)” and “weightweight percentage (% w / w)” are used interchangeably herein. Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, solution or product.

[0277] The terms “percent by volume”, “volume percentage (v%)” and “volumevolume percentage (% v / v)” are used interchangeably herein. Percent by volume refers to the percentage of a material on a volume basis as it is comprised in a composition, mixture, solution or product.

[0278] The term “16S rRNA” or “16S ribosomal RNA” means the rRNA constituting the small subunit of prokaryotic ribosomes. In bacteria, this sequence can be used to identify and characterize operational taxonomic units.

[0279] The term “ITS” or “Internal Transcribed Spacers” are regions within the ribosomal transcript that are excised and degraded during maturation. Their sequences can be used for phylogenetic analysis and / or identification of fungi or yeast. The terms moisturizer, a lotion or a body lotion refer to a low to mediumviscosity emulsion of oil and water, most often oil-in-water but possibly water-in-oil with the primary benefit in a skin care application to hydrate the skin or to reduce its water loss. Nearly all moisturizers contain a combination of emollients, occlusives, and humectants. Emollients, which are mainly lipids and oils, hydrate and improve the appearance of the skin. A wide variety of suitable emollients is known and maybe used herein (International Skin Care Ingredient Dictionary and Handbook, eds. Wenninger and McEwen, pp. 1656-61 , 1626, and 1654-55 (The Skin care, Toiletry, and Fragrance Assoc., Washington, D.C., 7thEdition, 1997) (referred to as “ ICI Handbook”) contains numerous examples of suitable materials). Occlusives such as petrolatum, lanolin and bee wax reduce transepidermal water loss by creating hydrophobic barrier over the skin. Humectants such as glycerol and urea able to attract water from the external environment and enhance water absorption from the dermis into the epidermis. In addition, the moisturizer formulations may contain emulsifiers to maintain stability of emulsions, and use thickeners to achieve desired viscosity and skin feel. A wide variety of other ingredients such as fragrances, dyes, preservatives, therapeutic agents, proteins and stabilizing agents are commonly added for other consumer preferred attributes.

[0280] The term “percent (%) sequence identity” or “percent (%) sequence similarity,” as used herein with respect to a reference sequence is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the residues in the reference polynucleotide sequence after optimal alignment of the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0281] As used herein, the term “probiotic” or “probiotic microorganism” are used interchangeably herein and refer to a live microorganism (including bacteria or yeasts for example) which, when administered (topically or orally) in sufficient amounts, beneficially affects the host organism, i.e. by conferring one or more demonstrable benefits, on the host organism. Whilst there are no lower or upper limits for probiotic use, it has been suggested that at least 106-1012, preferably at least 106-1010, preferably 108-109, cfu as a daily dose will be effective to achieve the beneficial effects in a subject.

[0282] A microbial “strain” as used herein refers to a microorganism (such as a bacterium or fungus) which remains genetically unchanged when grown or multiplied. The multiplicity of identical microbes is included.

[0283] As used herein, the term a “biologically pure strain” means a strain containing no other microbial strains in quantities sufficient to interfere with replication of the strain or to be detectable by normal techniques. “Isolated” when used in connection with the organisms and cultures described herein includes not only a biologically pure strain, but also any culture of organisms which is grown or maintained other than as it is found in nature.

[0284] In one aspect the skin cells described herein are mammalian skin cells, such as human or animal skin cells.

[0285] The term “sequence identity” or “sequence similarity” as used herein, means that two polynucleotide sequences, a candidate sequence and a reference sequence, are identical (i.e. 100% sequence identity) or similar (i.e. on a nucleotide-by-nucleotide basis) over the length of the candidate sequence. In comparing a candidate sequence to a reference sequence, the candidate sequence may comprise additions or deletions (i.e. gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for determining sequence identity may be conducted using the any number of publicly available local alignment algorithms known in the art such as ALIGN or Megalign (DNASTAR), or by inspection.

[0286] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0287] Unless defined otherwise 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.

[0288] Non-limiting examples of compositions and methods disclosed herein include: 1 . A skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization (protecting the skin against dehydration by maintaining, restoring and / or improving skin moisturization), and any one combination thereof. l b. A skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, treatment of a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization (protecting the skin against dehydration by maintaining, restoring and / or improving skin moisturization), and any one combination thereof. lc. The skin care composition of embodiment 1 or 1 b, wherein said skin barrier strengthening comprises administering the composition to the subject’s skin or scalp to strengthen the skin barrier of said subject. ld. The skin care composition of embodiment 1 or 1 b, wherein said improving skin barrier function comprises administering the composition to the subject’s skin or scalp to improve the skin barrier function of said subject. le. The skin care composition of embodiment 1 or 1 b, wherein said skin moisturization comprises administering the composition to the subject’s skin or scalp to moisturize the skin or scalp of said subject. lf. The skin care composition of embodiment 1 or 1 b, wherein said composition comprises an effective amount of a Bacillus subtilis fermentate extract at 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 relative to a total weight of said composition.

[0289] 1g. The skin care composition of embodiment 1 , wherein said composition comprises an effective amount of a Bacillus subtilis fermentate extract at 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 relative to a total volume of said composition.

[0290] 2. The skin care composition of embodiment 1 , wherein the Bacillus subtilis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus subtilis fermentate, a cell pellet extract obtained from a Bacillus subtilis fermentate, a whole broth fermentate extract obtained from a Bacillus subtilis fermentate, and any one combination thereof.

[0291] 3. The skin care composition of embodiment 1 , wherein the scalp or skin disorder is selected from the group consisting of an unbalanced ecoflora of the scalp, discomfort of the scalp, tinea versicolor, dry skin, irritated skin, and any one combination thereof. 4. The skin care composition of embodiment 1 , wherein said reducing of dandruff comprises administering the composition to the subject’s skin or scalp to reduce dandruff.

[0292] 4b. The skin care composition of embodiment 1 , wherein said reducing of said scalp or skin disorder comprises administering the composition to the subject’s skin or scalp to reduce said scalp or skin disorder.

[0293] 5. The skin care composition of any preceding embodiment, wherein the Bacillus subtilis fermentate extract, or fraction thereof, is obtained from a fermentate of a Bacillus subtilis selected from the group consisting of a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of 8. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0294] 6. The skin care composition of any preceding embodiment, further comprising one or more antidandruff active agents.

[0295] 7. The skin care composition of embodiment 1 , wherein the composition reduces growth of Malassezia species without affecting growth of bacteria selected from the group consisting of Staphylococcus hominis, Staphylococcus epidermis Cutibacteria acnes strains, and any one combinbation thereof.

[0296] 7b. The skin care composition of any preceding embodiment, wherein the composition reduces growth of Malassezia species without affecting growth of bacteria selected from the group consisting of Staphylococcus hominis, Staphylococcus epidermis , Cutibacteria acnes strains and any one combinbation thereof.

[0297] 8. The skin care composition of embodiment 1 , wherein said skin barrier strengthening and / or said improving skin barrier function and / or said skin moisturization occurs through fatty acid synthesis, cornification, small proline rich proteins synthesis, or any one combination thereof.

[0298] 8a. The skin care composition of any preceding embodiment, wherein said skin barrier strengthening occurs through fatty acid synthesis, cornification, small proline rich protein synthesis, or any one combination thereof.

[0299] 8b. The skin care composition of embodiment 8, wherein the fatty acid synthesis occurs through elongation of fatty acids.

[0300] 8c. The skin care composition of embodiment 8b, wherein the elongation of fatty acids occurs through increased expression of Elongation of Very Long Chain Fatty Acid (ELOVL) fatty acid elongase 7 (ELVOL7). 8d. The skin care composition of embodiment 8b, wherein the elongation of fatty acids occurs through increased expression of Elongation of Very Long Chain Fatty Acid (ELOVL) fatty acid elongase 7 (ELVOL7), when compared to the expression of said gene caused by a control composition that is idential to said skin care composition except for the absence of said effective amount of said Bacillus subtilis fermentate extract, or fraction thereof .

[0301] 8e. The skin care composition of embodiment 8b, wherein the fatty acid synthesis occurs through increased expression of Elongation of Very Long Chain Fatty Acid (ELOVL) fatty acid elongase 7 (ELVOL7).

[0302] 8f. The skin care composition of embodiment 8b, wherein the fatty acid synthesis occurs through increased expression of Elongation of Very Long Chain Fatty Acid (ELOVL) fatty acid elongase 7 (ELVOL7), when compared to the expression of said gene caused by a control composition that is idential to said skin care composition except for the absence of said effective amount of said Bacillus subtilis fermentate extract, or fraction thereof .

[0303] 8g. The skin care composition of embodiment 8, wherein the cornification occurs through an increased expression of a late cornified envelope 1c (LCE1 C) protein. 8h. The skin care composition of embodiment 8, wherein the small proline rich proteins synthesis occurs through an increased expression of small proline rich proteins (SPRR).

[0304] 8i. The skin care composition of embodiment 8, wherein the small proline rich proteins synthesis occurs through an increased expression of small proline rich proteins (SPRR), when compared to the expression of said gene caused by a control composition that is idential to said skin care composition except for the absence of said effective amount of said Bacillus subtilis fermentate extract, or fraction thereof.

[0305] 8j. The skin care composition of embodiment 8f, wherein the small proline rich protein is selected from the group consisting of SPRR2F, SPRR2E, SPRR2A, SPRR2D, and any one combination thereof.

[0306] 9. The skin care composition of any preceding embodiment further comprising an additional compound selected from the group consisting of a fragrance, an excipient, a preservative, a pH adjuster, an aqueous carrier, an alcohol carrier, an emollient, a solidification agent, a hydration agent, an emulsifier, a solubilizer, a surfactant, a thickening agent, a salt and any one combination thereof.

[0307] 10. Use of the skin care composition of any preceding embodiment in a cosmetic skin care product.

[0308] 10b. Use of an effective amount of the skin care composition of any preceding embodiment in a cosmetic skin care product.

[0309] 11 . A skin care product comprising the skin care composition of any preceding embodiment and one or more dermatologically or skin care acceptable component.

[0310] 11 b. A skin care product comprising a skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof. l l c. A skin care product comprising a skin care composition for use in reducing a skin or scalp disorder, the composition comprising an effective amount of a Bacillus subtilis fermentate, Bacillus subtilis fermentate extract, and / or fraction thereof, wherein said composition reduces and / or treats said skin or scalp disorder, and one or more dermatologically or skin care acceptable component. l l d. A skin care product comprising a skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the Bacillus subtilis is selected from the group consisting of a Bacillus subtilis having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO:1 ) of Bacillus subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a Bacillus subtilis having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO:2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a Bacillus subtilis suitable for use in the present invention also includes Bacillus subtilis having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO:3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a Bacillus subtilis having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO:7) of bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of 8. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0311] 12. The skin care product of embodiment 11 , wherein said 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% up to 10% of said skin care composition on a weight basis relative to a total weight of said skin care product.

[0312] 12b. The skin care product of embodiment 11 , wherein said 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% up to 10% of said skin care composition on a volume basis relative to a total volume of said skin care product.

[0313] 13. The skin care product of embodiment 11 , wherein the product is formulated for topical administration.

[0314] 13b. The skin care product of embodiment 11 , wherein the product is formulated for topical administration to the skin or scalp.

[0315] 14. The skin care product of any one of embodiment 11 -13, wherein the skin care product is selected from the group consisting of a lotion, a serum, a jelly, a cream, a gel, a hydrogel, an emulsion, a solid cosmetic, a mask, a patch, a shampoo, a loose or compact powder, a liquid suspension or solution, a spray solution and a stick.

[0316] 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 a Bacillus subtilis fermentate, and / or fraction thereof wherein the second skin care composition comprises at least an effective amount of at least one second active agent selected from antidandruff active agents, for topical administration.

[0317] 14c. A skin care product comprising a first skin care composition and a second skin care composition, wherein the first skin care composition an effective amount of a Bacillus subtilis fermentate, and / or fraction thereof; wherein the second skin care composition comprises at least an effective amount of at least one second active agent selected from antidandruff active agents, for topical administration, wherein the first skin care composition is formulated in at least one form selected from the group consisting of a gel, an emulsion, a hydrogel, a loose or compact powder, a liquid suspension or solution, or a spray solution.

[0318] 15. A method for providing at least one skin care benefit to a subject in need thereof, the method comprising topically administering a skin care composition or skin care product of any preceding embodiment to the subject’s skin or scalp.

[0319] 15b. A method for providing at least one skin care benefit to a subject in need thereof, the method comprising topically administering a skin care composition or skin care product comprising said composition to the skin or scalp of said subject, wherein said composition comprises an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0320] 15c. A method for providing at least one skin care benefit to a subject in need thereof, the method comprising topically administering a skin care composition or skin care product of any preceding embodiment to a subject in need thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, treatment of a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0321] 15d. A method for providing at least one skin care benefit to a subject in need thereof, the method comprising topically administering a composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, to a subject in need thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0322] 16. A method for reducing dandruff in a subject, the method comprising topically administering a skin care composition or skin care product of any preceding embodiment to the scalp of said subject.

[0323] 16b. A method for reducing dandruff in a subject, the method comprising topically administering a skin care composition or skin care product comprising said skin care composition to the scalp of said subject, wherein the skin care composition comprises an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof.

[0324] 16c. A method for reducing dandruff in a subject, the method comprising topically administering a skin care composition of any preceding embodiments or skin care product comprising said skin care composition to the scalp of said subject. 16d. A method for reducing dandruff in a subject, the method comprising topically administering a skin care composition or skin care product comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, to the scalp of said subject.

[0325] 16e. The method of embodiment 16b, wherein the skin care composition or skin care product is a selected from the group consisting of a lotion, a serum, a jelly, a cream, a gel, an emulsion, a solid cosmetic, a mask, a patch, and a stick comprising.

[0326] 16f. A method for reducing a scalp or skin disorder in a subject, the method comprising topically administering a skin care composition or skin care product comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, to the scalp or skin of said subject.

[0327] 16g. A method for reducing a scalp or skin disorder in a subject, the method comprising topically administering a skin care composition or skin care product comprising said skin care composition to the scalp of said subject, wherein the skin care composition comprises an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof.

[0328] 16h. A method for reducing a scalp or skin disorder in a subject, the method comprising topically administering a skin care composition or skin care product comprising said skin care composition to the scalp of said subject, wherein the skin care composition comprises an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the scalp or skin disorder is selected from the group consisting of a dandruff condition of the scalp (seborrheic dermatitis), unbalanced ecoflora of the scalp, discomfort of the scalp, tinea versicolor, dry skin, irritated skin, and any one combination thereof.

[0329] 16i. The method of embodiment 16, 16b, 16c, 16d, 16e, 16f, or 16f , wherein the fermentate extract reduces growth of Malassezia species.

[0330] 17. A method for skin barrier strengthening in a subject, the method comprising topically administering a skin care composition or skin care product of any preceding embodiment, to the skin or scalp of said subject. 17b. A method for skin barrier strengthening in a subject, the method comprising topically administering a skin care composition or skin care product comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, to the skin or scalp of said subject.

[0331] 17c. A method for improving skin barrier function in a subject, the method comprising topically administering a skin care composition or skin care product of any preceding embodiment, to the skin or scalp of said subject.

[0332] 17d. A method for improving skin barrier function in a subject, the method comprising topically administering a skin care composition or skin care product comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, to the skin or scalp of said subject.

[0333] 18. A method for skin moisturization in a subject, the method comprising topically administering a skin care composition or skin care product of any preceding embodiment to the skin or scalp of said subject.

[0334] 18b. A method for skin moisturization in a subject, the method comprising topically administering a skin care composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, to the skin or scalp of said subject.

[0335] 19. The method of any one of embodiment 15-embodiment 18, wherein the Bacillus subtilis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus subtilis fermentate, a cell pellet extract obtained from a Bacillus subtilis fermentate, a whole broth fermentate extract obtained from a Bacillus subtilis fermentate, and any one combination thereof.

[0336] 19b. The method of any one of embodiment 15-embodiment 19, wherein the Bacillus subtilis fermentate extract is obtained from a fermentate of a Bacillus subtilis selected from the group consisting of a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of 8. subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a 8. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of 8. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0337] 19c. The method, the skin care composition for use, or the skin care product of any one of the preceding embodiments, wherein the skin care product or skin care composition is selected from the group consisting of a lotion, a serum, a jelly, a cream, a gel, an emulsion, a solid cosmetic, a mask, a patch, a shampoo, and a stick comprising at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% up to 10% of said skin care composition on a weight basis relative to a total weight of said skin care product. 20. A Bacillus subtilis strain variant fermentate, Bacillus subtilis strain variant fermentate extract, or fraction of said variant fermentate, of a B. subtilis strain variant, wherein said Bacillus subtilis strain variant is derived from a parental B. subtilis by knocking out at least one sporulation gene of said parental B. subtilis strain, wherein said variant fermentate, said variant fermentate extract, or said fraction of said variant fermentate, has increased activity when compared to the activity of a fermentate, a fermentate extract, or fraction thereof derived from said parental B. subtilis strain, wherein said activity is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0338] 20b. A Bacillus subtilis strain variant fermentate, Bacillus subtilis strain variant fermentate extract, or fraction of said variant fermentate, of a B. subtilis strain variant, wherein said Bacillus subtilis strain variant is derived from a parental B. subtilis by knocking out a SpollE sporulation gene of said parental B. subtilis strain, wherein said variant fermentate, said variant fermentate extract, or said fraction of said variant fermentate, has increased activity when compared to the activity of a fermentate, a fermentate extract, or fraction thereof derived from said parental B. subtilis strain, wherein said activity is selected from the group consisting of reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0339] 21 . A method for increasing the activity of a Bacillus subtilis strain fermentate 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 fermentate extract for providing at least one skin care benefit selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof; (b) modifying said parent Bacillus subtilis strain by knocking out at least one sporulation gene from the genome of said Bacillus subtilis strain, thereby producing a variant strain that is unable to form spores; and, (c) producing a fermentate extract of said Bacillus subtilis variant strain, wherein said fermentate extract of said Bacillus subtilis variant strain has an increased activity for providing at least one skin care benefit selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof, when compared to the activity of the fermentate extract of said parent Bacillus subtilis strain.

[0340] 21 b. A method for increasing the activity of a Bacillus subtilis strain fermentate 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 fermentate extract for providing at least one skin care benefit selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof; (b) modifying said parent Bacillus subtilis strain by knocking out a SpollE sporulation gene from the genome of said Bacillus subtilis strain, thereby producing a variant strain that is unable to form spores; and, (c) producing a fermentate extract of said Bacillus subtilis variant strain, wherein said fermentate extract of said Bacillus subtilis variant strain has an increased activity for providing at least one skin care benefit selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof, when compared to the activity of the fermentate extract of said parent Bacillus subtilis strain.

[0341] 22. A skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises at least one active agent of less than 3 Kda. 23. A skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises between 0.1 % to 100% of at least one active agent of less than 3 Kda on a weight basis relative to a total weight of said effective amount.

[0342] 24. A skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises between 0.1 % to 100% of at least one active agent of less than 3 Kda on a volume basis relative to a total volume of said effective amount.

[0343] 25. The skin care composition of embodiments 22-24, wherein said at least one active agent is bacilysin.

[0344] 26. A skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, growth inhibition of Malassezia, growth of Malassezia species without affecting growth of Staphylococcus hominis, Staphylococcus epidermis and Cutibacteria acnes strains and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises at least one active agent of less than 3 Kda.

[0345] 27. A skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, growth inhibition of Malassezia, growth of Malassezia species without affecting growth of Staphylococcus hominis and Cutibacteria acnes strains and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises between 0.1 % to 100% of at least one active agent of less than 3 Kda on a volume basis relative to a total volume of said effective amount.

[0346] 28. A skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, growth inhibition of Malassezia, growth of Malassezia species without affecting growth of Staphylococcus hominis and Cutibacteria acnes strains and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises between 0.1 % to 100% of at least one active agent of less than 3 Kda on a weight basis relative to a total weight of said effective amount.

[0347] 29. The skin care composition of embodiments 26-28, wherein said at least one active agent is bacilysin.

[0348] 30. A skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate 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 one combination thereof. 31 . A skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus subtilis fermentate extract, or fraction thereof, wherein the at least one skin care benefit is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, and wherein the effective amount of said Bacillus subtilis fermentate extract comprises an active agent of less than 3 Kda.

[0349] 32. The skin care composition of any one of embodiments 22-31 , wherein the fermentate extracts, or fractions thereof, includes fermentate extracts, or fractions thereof, from the microorganism Bacillus subtilis, wherein said Bacillus subtilis is selected from a Bacillus subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of Bacillus subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a bacterial strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0350] EXAMPLES

[0351] In the following Examples, unless otherwise stated, parts and percentages are by weight and degrees are Celsius. It should be understood that these Examples, while indicating embodiments of the disclosure, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can make various changes and modifications of the disclosure to adapt it to various usages and conditions. Such modifications are also intended to fall within the scope of the appended claims.

[0352] The following abbreviations in the specification correspond to units of measure, techniques, properties, or compounds as follows: “sec” or “s” means second(s), “min” means minute(s), “h” or “hr” means hour(s), “p.L” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “ppm” means part(s) per million, “wt” means weight, “wt%” means weight percent, “g” means gram(s), “mg” means milligram(s), “pg” means microgram(s), “ng” means nanogram(s), “cone.” means concentration, “Trt” means treatment. EXAMPLE 1

[0353] Bacillus subtilis strains

[0354] To obtain a variety of strains of Bacillus, various fermented food and beverages were treated by heating at 80°C for 20 minutes. After heat treatment, an aliquot was spread on tryptic soy broth agar plates. Each liter of tryptic soy broth contains 17 grams of trypticase peptone, 3 grams of soytone, 5 grams of sodium chloride, 2.5 grams, dipotassium phosphate, 2.5 grams dextrose. The phylogenetic identity of each Bacillus strain was determined by sequencing the 16S region with primer set 8F (AGA GTT TGA TYM TGG CTC; SEQ ID NO: 4) and 515F (GTG CCA GCM GCC GCG GTA AA; SEQ ID NO: 5), where “M” is nucleotide Adenine or Cytosine. The 16S region was amplified with primer set 8F and 1492R (CGG TTA CCT TGT TAC GAC TT; SEQ ID NO: 6).

[0355] Bacillus suitable for use in the present invention includes, but is not limited to, a Bacillus selected from the group consisting of a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of 8. subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149857, a 8. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969 (The genome-wide Average Nucleotide Identity of Bacillus subtilis PC01 and Bacillus subtilis subsp. inaquosorum is 98.75% so it can also be referred to as a Bacillus inaquosorum type strain), a 8. subtilis strain having a 16S ribosomal RNA sequence displaying 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 (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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% sequence similarity to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

[0356] In addition, Bacillus subtilis 168 (ATCC23857) having a 16S ribosomal RNA sequence of SEQ ID NO: 8, was used for comparison.

[0357] EXAMPLE 2 Fermentates of Bacillus subtilis

[0358] Bacillus subtilis strains were grown in SUM (Soyptone Urea Micronutrient) medium. Each liter of SUM medium (1 .0 X strength) consists of 10 g soytone, 75 g glucose, 3 ml of dipotassium phosphate (1 M), 3.6 g urea, and 10 ml of micronutrient stock. Each liter of the micronutrient stock contained 1 ,47g Sodium Citrate 2H2O, 1.47 CaCI2-2 H2Q, 0.4 FeSO4 -7 H2Q, 0.1 g MnSO4- H2Q, 0.1 g ZnSO4- H2Q, 0.05 g CuCI2.2 H2Q, 0.1 g C0CI2 6 H2Q, and 0.1 g Na2MoO4-2 H2Q. The SUM medium was adjusted to pH 7.3. Generation of B. subtilis fermentate was carried out in 125 ml flat bottom shake flasks with 25 ml medium. The flasks were placed in a platform shaker with a constant speed of 200 rpm at 32 °C. The overnight fresh seed culture was used as inoculum and the starting OD was 0.125 at 600 nm. The culture was allowed to grow for 48 hours. Bacillus subtilis can also be grown in TSB (Tryptic Soy Broth) medium supplemented with 1 % glucose (TSG). Each liter of TSG medium (1 .0 X strength) consists of 17 g tryptone, 3 g soytone, 5 g sodium chloride, 10 g glucose, and 2.5 g of dipotassium phosphate. The TSG medium can be adjusted to pH 7.3. Even though SUM medium was used for growth medium of B. subtilis as described herein, B. subtilis strains can be grown in any suitable media that can support growth. In addition, the incubation time may vary depending on the growth medium or method of growth. It is understood that the fermentate can be collected at different hours of incubation and still produce a fermentate that contains an effective amount of a B. subtilis fermentate extract.

[0359] EXAMPLE 3

[0360] Bacillus subtilis fermentate extracts

[0361] Extracts of the Bacillus subtilis fermentates were obtained as described below.

[0362] Cell free supernatants of Bacillus subtilis fermentate

[0363] Cells were removed from the Bacillus subtilis fermentate by pelleting cells by centrifugation at 4,000 g for 20 min. The cell free supernatant (fermentate extract) obtained was sterilized by passing through a 0.2 pM filter. Cell pellet extracts of Bacillus subtilis fermentate

[0364] A cell pellet extract from the Bacillus subtilis fermentate is obtained by pelleting cells (centrifuge at 4,000 to 8,000 x g) and pouring off the supernatant, to leave behind the portion of cell pellet. The pellet is resuspended in acidic water (1 / 10 v / v) to a pH 2.5 - 4.0, vortexed, pelleted by centrifugation (4,000 to 8,000 x g) and the extract liquid is passed through a 0.2 pM filter.

[0365] Starting from a cell pellet has the advantage that the volume of the resuspension liquid (extract volume) can be determined to produce an effective amount of the active extract and / or further concentrated to produce an effective amount of the active extract. Furthermore, resuspending the pellet in water or nonfermentation broth liquids, results in reducing or eliminating the non-active ingredients of the fermentation broth which may interfere with the actives of the cell pellet extract.

[0366] Combination of cell free supernatant and cell pellet extracts of Bacillus subtilis fermentate

[0367] Bacillus subtilis fermentate extracts can also be produced by combining the cell free supernatant fraction described above with the cell pellet extract described above.

[0368] Whole broth Bacillus subtilis fermentate extracts

[0369] To obtain a whole broth fermentate extract, the Bacillus subtilis fermentate extract is prepared by first adjusting the pH of the fermentate (total fermentation broth -Example 2) to a pH 2.5 - 4.0 prior to pelleting out the insoluble cellular matter and filtering the supernatant through a 0.2 pM filter to yield a pH adjusted cell free supernatant. Alternatively, the Bacillus subtilis whole broth fermentate extract is prepared by first adjusting the pH of the fermentate (total fermentation broth) to an alkaline pH prior to pelleting out the insoluble cellular matter and optionally filtering supernatant through a 0.2 pM filter to yield a cell free supernatant (also referred to as “whole broth fermentate extract”).

[0370] EXAMPLE 4

[0371] Growth Inhibition of Malassezia species by 5. subtilis fermentate extracts.

[0372] The ability of B. subtilis fermentate extracts to inhibit the growth of Malassezia species was evaluated. B. subtilis G01 (CBS149857), B. subtilis N01 (CBS149971 ), B. subtilis K01 (CBS149972), and B. subtilis PC01 (CBS149969) were grown in SUM (Soyptone Urea Micronutrient) medium. Each liter of SUM medium (1 .0 X strength) consists of 10 g soytone, 75 g glucose, 3 ml of dipotassium phosphate (1 M), 3.6 g urea, and 10 ml of micronutrient stock. Each liter of the micronutrient stock contained 1.47g Sodium Citrate 2H2O, 1.47 CaCl2-2 H2O, 0.4 FeSO4 -7 H2O, 0.1 g MnS04- H2O, 0.1 g ZnSO4- H2O, 0.05 g CuCI2.2 H2O, 0.1 g C0CI2 6 H2O, and 0.1 g Na2MoO4 2 H2O. The SUM medium was adjusted to pH 7.3. SUM medium at 0.5 x strength was obtained by diluting the SUM medium by half with purified water. Generation of B. sub tilis fermentate was carried out in 125 ml flat bottom shake flasks with 25 ml SUM medium at 0.5x strength. The flasks were placed in a platform shaker with a constant speed of 200 rpm at 32°C. The overnight fresh seed culture was used as inoculum and the starting OD was 0.125 at 600 nm. After growth for 48 hours, the cell free supernatant (fermentate extract) was obtained by centrifugation and filtersterilization of the fermentate. For comparison purpose, Bacillus subtilis 168 (ATCC23857) was used and grown under the same conditions.

[0373] To determine the growth inhibition-activity of the fermentate extract of B. subtilis, a growth inhibition assay was performed using Malassezia furfur (M. furfur) as the target organism in a zone inhibition assay using agar plate. M. furfur ATCC 14521 was cultured by inoculating 1 :100 from frozen stock into mDixon media containing 500 pg / mL chloramphenicol and incubated at 32 °C in an orbital shaker at 250 rpm for 2 days before the growth inhibition-activity test. Each liter of mDixon medium contains 36 g malt extract, 20 g desiccated oxbile, 10 ml Tween-40, 6 g peptone, 2 ml glycerol, and 2 ml oleic acid. The medium pH was adjusted to 6. Fresh M. furfur cells were suspended in mDixon broth to obtain an optical density equivalent to an OD of 0.4. An aliquot of 100 pl was used to seed an agar plate evenly. After drying, 5 pl of fermentate extract of B. subtilis strains were spotted directly on the top of the agar. The plate was incubated at 32°C for 48 hours. The presence of a clear zone is indicative of growth inhibition and the size of the inhibition zone was used as an indicator of the efficacy of the fermentate extract. The size of the zone was measured based on the diameter in millimeter (mm). Results are shown in Table 1 .

[0374] Table 1 . Growth inhibition assay of Malassezia using zone inhibition assay with fermentate extracts of different Bacillus strains.

[0375] As shown in Table 1 , the fermentate extract obtained from the Bacillus subtilis strain 168 (ATCC 23857) did not show any growth inhibition of Malassezia while fermentate extracts of B. subtilis PC01 and B. subtilis N01 had good growth inhibiton activity. The fermentate extracts of B. subtilis strains G01 and K01 had a larger zone of inhibition, also indicating a high efficacy for growth inhibition of Malassezia.

[0376] Human skin is colonized by a diverse microbiota (Byrd, A., Belkaid, Y. & Segre, J. The human skin microbiome. Nat Rev Microbiol 16, 143-155; 2018). Staphylococcus hominis and Cutibacterium (formerly known as Propionibacterium) are two of the dominant commensal species. In order to evaluate the specificity of the activity against other skin microbes by fermentate extracts of B. subtilis G01 , Staphylococcus hominis ATCC 27844, Staphylococcus epidermidis ATCC 12228 and Cutibacteria acnes K2 strains were used as target organisms in a zone inhibition assay. Direct spotting of the 5 pl of the fermentate extract of B. subtilis G01 did not result in visible clearing zones for both organisms. This result indicated that the fermentate extract generated from B. subtilis G01 had high specificity towards yeast skin Malassezia without affecting growth of Staphylococcus hominis, Staphylococcus epidermidis and Cutibacteria acnes strains. EXAMPLE 5

[0377] Improvement of skin barrier function using B. subtilis fermentate extract.

[0378] The ability of B. subtilis fermentate extracts to improve the skin barrier & skin barier function was evaluated. B. subtilis G01 strain (CBS149857) was grown in TSB (Tryptic Soy Broth) medium supplemented with 1 % glucose (TSG). Each liter of TSG medium (1.0 X strength) consists of 17 g tryptone, 3 g soytone, 5 g sodium chloride, 10 g glucose, and 2.5 g of dipotassium phosphate. The TSG medium was adjusted to pH 7.3. Generation of B. subtilis fermentate was carried out in 125 ml flat bottom shake flasks with 25 ml medium. The flasks were placed in a platform shaker with a constant speed of 200 rpm at 32°C. The overnight fresh seed culture was used as inoculum and the starting OD was 0.125 at 600 nm. After growth for 24 hours, the cell free supernatant (fermentate extract) was obtained by centrifugation and filter-sterilization.

[0379] Normal human epidermal keratinocytes (NHEK) isolated from a single adult donor were thawed in Keratinocyte Growth Media 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 pg / mL), hydrocortisone (0.33 pg / mL), epinephrine (0.39 pg / mL), transferrin (10 pg / mL) and calcium chloride (0.06 mM). NHEK were subsequently cultured in a T75 flask containing KGM2 to achieve 75-80% confluency in a 37°C incubator supplied with 5% CO2 and 95% humidity. Following reaching desired confluency, NHEK were removed from the T75 flask using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK were resuspended in KGM2, counted using a hemocytometer and seeded onto transwell inserts (6.5 mm insert; 0.4 pm polyester membrane; tissue culture treated, polystyrene 24 well plate). NHEK were incubated in a 37°C incubator supplied with 5% CO2 and 95% humidity until cells reached 100% confluency on transwell membrane. At that point, the media was aspirated and NHEK were subsequently grown in KGM2 supplemented with 1 .5 mM calcium chloride instead of 0.06 mM for the remainder of the experiment. To the top well, B. subtilis G01 fermentate extract was added such that the final fermentate extract concentration was 0.1 %. Following addition of the fermentate extract, the transepidermal electrical resistance (TEER) was measured every day using an epithelial volt / ohm meter (EVOM). Media and fermentate extract were refreshed every 2 days. Results are shown in Table 2. Table 2. B. subtilis G01 fermentate extract increases Transepidermal Electrical Resistance (TEER).

[0380] The results in Table 2 indicate that compared to untreated (no fermentate extract) control, addition of 0.1 % B. subtilis G01 fermentate extract resulted in a higher percent increase in TEER values, which is an indication of improved skin barrier function and strengthening of the skin barrier.

[0381] EXAMPLE 6

[0382] Improvement of skin barrier function using fractions of B. subtilis fermentate extract.

[0383] The ability of specific molecular weight fractions of B. subtilis fermentate extract to improve the skin barrier function was evaluated. Fermentate extract from B. subtilis G01 strain (CBS149857) was generated using the same method as described in Example 5. Following centrifugation and filter sterilization, the cell free supernatant (fermentate extract) was then fractionated by molecular weight by passing the supernatant through a series of molecular weight cut-off filters in the following progression: 100 kilodalton (kDa), 50 kDa, 30 kDa and 10 kDa. Fermentate extracts that did not pass through the filters were saved and referred to as retentates (R) and fermentate extract that passed through the filters were saved and referred to as flow throughs (FT). Each B. subtilis G01 molecular weight retentate and flow through were tested for the ability to improve skin barrier function compared to an untreated (no fermentate extract) control using NHEK as performed in Example 5. Results are shown in Table 3A.

[0384] Table 3A. Various B. subtilis G01 molecular weight fermentate extract fractions increase Transepidermal Electrical Resistance (TEER).

[0385] The results in Table 3A indicate that compared to untreated (no fermentate extract) control, addition B. subtilis G01 fermentate extract (cell free supernatant) passed through a 100 kDa, 50 kDa, and 30 kDa molecular weight cut-off filter at a concentration of 0.1 % in TEER assay, had a higher percent increase in TEER values, which is an indication of improved skin barrier function and strengthening of the skin barrier. We further evaluated the 10 kDa flow through for its ability to improve the skin barrier function by first concentrating the 10 kDa flow through ten times (1 OX), prior to adding it to the TEER assay. Briefly, an aliquot of the 10 kDa flow through fermentate extract was centrifuged under vacuum until the final volume achieved 10 % of the starting volume. This fraction is referred to as 10 kDa flow through

[0386] 10X. The 10 kDa flow through 10X sample was then tested for the ability to improve skin barrier function compared to an untreated (no fermentate extract) control in a dose dependent manner such that the final assay concentrations were 0.05, 0.1 , 0.2 and 0.4 % of the starting concentrated fermentate extract. Accounting for the concentration factor, the final assay concentrations, 0.05, 0.1 , 0.2 and 0.4 % represent 0.5, 1 , 2, and 4 % of the unconcentrated fermentate extract. The results are shown in Table 3B.

[0387] Table 3B. The < 10 kPa flow through 10X molecular weight fermentate extract fraction increases Transepidermal Electrical Resistance (TEER).

[0388] The results in Table 3B indicate that compared to the untreated (no fermentate extract) control, the concentrated form of the less than 10 kDa B. subtilis G01 fermentate extract can increase TEER values, which is an indication of improved skin barrier functioning.

[0389] EXAMPLE 7.

[0390] Improvement of skin barrier function through a heat sensitive B. subtilis fermentate extract.

[0391] The ability of heat sensitive B. subtilis fermentate extract to improve the skin barrier function was evaluated. Fermentate extract (cell free supernatant) from B. subtilis G01 strain (CBS 149857) fermentate was generated using the same method as described in Example 5. Prior to using in the assay, B. subtilis G01 fermentate extract was incubated at 100°C for 10 minutes. Subsequently, B. subtilis G01 non-heat treated and heat treated fermentate extracts were tested for the ability to improve skin barrier function using NHEK as performed in Example 5. (Table 4). Results are compared to an untreated (no fermentate extract) control.

[0392] Table 4. B. subtilis G01 component that increases the keratinocyte barrier is heat sensitive The results in Table 4 demonstrate that after heating at 100°C, the B. subtilis G01 fermentate extract was unable to increase the keratinocyte barrier.

[0393] EXAMPLE 8.

[0394] Improvement of skin barrier function using fermentate extracts of multiple strains of B. subtilis

[0395] The ability of fermentate extracts from multiple strains of B. subtilis to improve the skin barrier function was evaluated. Fermentate extracts (cell free supernatants) of B. subtilis G01 (CBS149857), B. subtilis 168 (ATCC23857), B. subtilis N01 (CBS149971 ) and B. subtilis K01 (CBS149972) strains were generated using the same method as described in Example 5. Each B. subtilis fermentate extract was tested for the ability to improve skin barrier function compared to an untreated (no fermentate extract) control using NHEK as performed in Example 5.

[0396] Table 5. Fermentate extracts (cell free supernatent) of B. subtilis G01 and B. subtilis N01 strains increase Transepidermal Electrical Resistance (TEER).

[0397] The results shown in Table 5 demonstrates that fermentate extracts (cell free supernatant) of 8. subtilis G01 and B. subtilis N01 increase Transepidermal Electrical Resistance (TEER), which is an indication of improved skin barrier function and strengthening of the skin barrier. EXAMPLE 9.

[0398] Strengthening of the skin barrier by B. subtilis fermentate extract through fatty acid synthesis

[0399] The mechanism by which fermentate extracts of B. subtilis affects the keratinocyte barrier was investigated. Normal human epidermal keratinocytes (NHEK) isolated from a single adult donor were thawed in Keratinocyte Growth Media 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 pg / mL), hydrocortisone (0.33 pg / mL), epinephrine (0.39 pg / mL), transferrin (10 pg / mL) and calcium chloride (0.06 mM). NHEK were subseguently cultured in a T75 flask containing KGM2 to achieve 75-80% confluency in a 37°C incubator supplied with 5% CO2 and 95% humidity. Following reaching desired confluency, NHEK were removed from the T75 flask using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK were resuspended in KGM2, counted using a hemocytometer and seeded into 96 well plates. Fermentate extract (cell free supernatant) from B. subtilis G01 (CBS149857) was generated as described in Example 5. Cells were incubated overnight in a 37°C incubator supplied with 5% CO2 and 95% humidity and then stimulated with B. subtilis G01 fermentate extract for 18 hours. Following stimulation with B. subtilis G01 fermentate extract, RNA was isolated from the NHEK using the RNAqueous Micro Kit. The RNA was further processed using the Illumina stranded mRNA prep kit such to extract the mRNA from all other RNA types. Individual mRNA libraries were normalized to 2 nM, pooled and 4% phiX added to the pooled mRNA library. One (1 ) nM of the pooled library was loaded onto either a P1 , P2 or P3 Illumina chip and then sequenced using primer A (SEQ ID NO: 9) and primer B ( SEQ ID NO: 10) using either the Illumina NextSeq 1000 or Illumina NextSeq 2000 instrument. Results are shown in Table 6. Table 6. Fermentate extract of B. subtilis G01 increases the expression of Elongation of Very Long Chain Fatty Acid (ELOVL) fatty acid elonqase 7 (ELOVL7) in NHEK.

[0400] The results presented in Table 6 demonstrate that the fermentate extract (cell free supernatant) of B. subtilis G01 increased the expression of the ELVOL7 gene (Gene ID 79993) in NHEK indicating that B. subtilis G01 strengthens the keratinocyte barrier (skin barrier) through elongation of fatty acids and therefore suitable use in strengthening the skin barrier, skin moisturization, reducing dandruff, and reducing skin and scalp disorders.

[0401] EXAMPLE 10.

[0402] Strengthening of the skin barrier by fermentate extract of B. subtilis through cornification

[0403] The mechanism by which B. subtilis fermentate extract affects the keratinocyte barrier was analyzed. Normal human epidermal keratinocytes (NHEK) isolated from a single adult donor were thawed in Keratinocyte Growth Media 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 pg / mL), hydrocortisone (0.33 pg / mL), epinephrine (0.39 pg / mL), transferrin (10 pg / mL) and calcium chloride (0.06 mM). NHEK were subsequently cultured in a T75 flask containing KGM2 to achieve 75- 80% confluency in a 37°C incubator supplied with 5% CO2 and 95% humidity. Following reaching desired confluency, NHEK were removed from the T75 flask using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK were resuspended in KGM2, counted using a hemocytometer and seeded into 96 well plates. Fermentate extract (cell free supernatant) from B. subtilis G01 (CBS149857) was generated as described in Example 5. Cells were incubated overnight in a 37°C incubator supplied with 5% CO2 and 95% humidity and then stimulated with B. subtilis G01 fermentate extract for 18 hours. Following stimulation with B. subtilis G01 fermentate extract, RNA was isolated from the NHEK using the RNAqueous Micro Kit. The RNA was further processed using the Illumina stranded mRNA prep kit such to extract the mRNA from all other RNA types. Individual mRNA libraries were normalized to 2 nM, pooled and 4% phiX added to the pooled mRNA library. 1 nM of the pooled library was loaded onto either a P1 , P2 or P3 Illumina chip and then sequenced using primer A (SEQ ID NO: 9) and primer B ( SEQ ID NO: 10) using either the Illumina NextSeq 1000 or Illumina NextSeq 2000 instrument. Results are sown in Table 7.

[0404] Table 7. Fermentate extract (cell free supernatant) of B. subtilis G01 increases the expression of late cornified envelope 1 c (LCE1 C) in NHEK

[0405] The results presented in Table 7 demonstrate that the fermentate extract (cell free supernatant) of B. subtilis G01 increased the expression of the LCE1 C gene (Gene ID 353133) in NHEK indicating that B. subtilis G01 strengthens the keratinocyte barrier (skin barrier) through increased cornification and is therefore suitable for use in strengthening the skin barrier, skin moisturization, reducing dandruff, and reducing skin and scalp disorders.

[0406] EXAMPLE 11.

[0407] Improvement of skin barrier by B. subtilis G01 fermentate extract through synthesis of small proline rich proteins

[0408] The mechanism by which B. subtilis fermentate extract affects the keratinocyte barrier was analyzed. Normal human epidermal keratinocytes (NHEK) isolated from a single adult donor were thawed in Keratinocyte Growth Media 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 pg / mL), hydrocortisone (0.33 pg / mL), epinephrine (0.39 pg / mL), transferrin (10 pg / mL) and calcium chloride (0.06 mM). NHEK were subsequently cultured in a T75 flask containing KGM2 to achieve 75- 80% confluency in a 37°C incubator supplied with 5% CO2 and 95% humidity. Following reaching desired confluency, NHEK were removed from the T75 flask using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK were resuspended in KGM2, counted using a hemocytometer and seeded into 96 well plates. Fermentate extract (cell free supernatant) from B. subtilis G01 (CBS149857) was generated as described in Example 5. Cells were incubated overnight in a 37°C incubator supplied with 5% CO2 and 95% humidity and then stimulated with B. subtilis G01 fermentate extract for 18 hours. Following stimulation with B. subtilis G01 fermentate extract, RNA was isolated from the NHEK using the RNAqueous Micro Kit. The RNA was further processed using the Illumina stranded mRNA prep kit such to extract the mRNA from all other RNA types. Individual mRNA libraries were normalized to 2 nM, pooled and 4% phiX added to the pooled mRNA library. 1 nM of the pooled library was loaded onto either a P1 , P2 or P3 Illumina chip and then sequenced using primer A (SEQ ID NO: 9) and primer B ( SEQ ID NO: 10) using either the Illumina NextSeq 1000 or Illumina NextSeq 2000 instrument. Results are sown in Table 8.

[0409] Table 8. Fermentate extract (cell free supernatant) of B. subtilis G01 increases the expression of small proline rich protein (SPRR) 2F, SPRR2E, SPRR2A and SPRR2D in NHEK.

[0410] The results presented in Table 8 demonstrate that B. subtilis G01 increased 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 B. subtilis G01 strengthens (increases) the keratinocyte barrier through small proline rich proteins synthesis, and is therefore suitable for use in reducing scalp disorders such as dandruff and seborrheic dermatitis, as well as for skin barrier strengething and skin moisturization.

[0411] EXAMPLE 12

[0412] Knocking out B. subtilis sporulation gene spo / / E further enhances the ability of B. subtilis fermentate extracts to improve the skin barrier function.

[0413] B. subtilis strains contain genes associated with sporulation such as SpollE (SEQ ID NO: 11 ; Barak et al. 1996, Molecular Microbiology 19(5), 1047-1060). In one aspect, elimination of spollE gene is desired, especially for B. subtilis production strains.

[0414] The ability of B. subtilis G01 sporulation spollE knockout (referred to as B. subtilis G01_spo, CBS151098) fermentate extract to improve the skin barrier function was evaluated.

[0415] A CRISPR-Cas9 plasmid (referred to as pMY1 -3) was constructed to delete the spollE locus from B. subtilis G01 (CBS147469) and, thereby, inhibits its ability to produce spores. The pMY1 -3 plasmid comprised a Streptococcus pyogenes Cas9 endonuclease expression cassette as well as an editing template insert (SEQ ID NO: 12), containing the B. subtilis G01 homologous regions and the sgRNA (SEQ ID NO: 13), and the kanamycin resistance cassette. The pMY1-3 plasmid was electroporated into Endura™ Electrocom petent Cells (Biosearch Technologies). The assembled construct was verified via Sanger sequencing. Rolling-circle amplification (RCA) was used to amplify pMY1 -3 using the GenomiPhi HY Ready-To-Go DNA Amplification Kit (Cytiva).

[0416] The B. subtilis G01 (CBS147469) was modified to contain the competence plasmid pBL.comK, as described in WO2019 / 40423, and a deletion of the spollE gene. Briefly, pBL.comK was transformed into B. subtilis G01 using natural competence and with spectinomycin as the resistance marker. Then, the pBL.comK-containing strain was made competent, as described in WO2021 / 146411 , and subsequently transformed with plasmid pMY1 -3. A volume of 200 pL competent cells was mixed with 50 pL pMY1 -3 RCA product and 1 mL Luria-Bertani (LB) broth and then 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 for cells transformed with the Cas9 plasmid pMY1 -3. The isolates were screened for the deletion of spollE using Q5® PCR with primer BSspo-check-del-F (SEQ ID NO: 14) and primer BSspo-check-del-R (SEQ ID NO: 15). Colonies with the spollE deletion ( spollE) produced a PCR product of 2448 bp, whereas the wild-type strain containing the intact spollE gene produced a PCR product of 4932 bp. Deletions were confirmed via Sanger sequencing, and confirmed mutant isolates were stored as glycerol stocks at -80°C.

[0417] The isolates were cured of pBL.comK and pMY1 -3 by culturing them in TSA containing no antibiotics for three days, passaging once every 24 hours, and then plating onto plain tryptic soy agar (TSA). Colonies were selected from the TSA and patched onto LB plates containing no antibiotics, LB containing 20 ppm kanamycin, and LB plates containing 100 ppm spectinomycin. Cured isolates grew on plain LB and not LB containing kanamycin or spectinomycin.

[0418] Following genotypic confirmation of the spollE deletion, phenotypic analysis using heat treatment was conducted to confirm the loss of sporulation ability. B. subtilis G01 was grown in Difco sporulation medium (DSM) to promote the formation of spores (Nicholson and Setlow, 1990). The wild-type and spollE clones were freshly plated on TSA, and single colonies were inoculated in DSM and incubated at 32°C and 200 RPM for three days. Two 100-pL aliquots were removed from each culture and transferred to 1 ,5-mL microcentrifuge tubes. The test aliquot was heated at 90°C for 15 minutes, while the control aliquot remained at room temperature (RT). The entirety of the 100-pL aliquots was then plated onto TSA and incubated overnight at 32°C. Sporulation-positive isolates, such as the wild-type B. subtilis G01 , displayed growth at both room temperature (RT) and the 90°C treatment. Sporulation-negative isolates displayed growth at RT but not at 90°C. Sporulation-negative clones (also referred to as B. subtilis G01_spo strain) were stored as glycerol stocks at -80°C. B. subtilis G01 (CBS147469, also referred to as B. subtilis G01 wild type (WT)), and B. subtilis G01_spo strains were grown in TSB (Tryptic Soy Broth) medium supplemented with 1 % glucose (TSG). Each liter of TSG medium (1 .0 X strength) consists of 17 g tryptone, 3 g soytone, 5 g sodium chloride, 10 g glucose, and 2.5 g of dipotassium phosphate. The TSG medium was adjusted to pH 7.3. Generation of B. subtilis G01 WT and KO fermentate was carried out in 125 ml flat bottom shake flasks with 25 ml TSG. The flasks were placed in a platform shaker with a constant speed of 200 rpm at 32°C. The overnight fresh seed culture was used as inoculum and the starting OD was 0.125 at 600 nm. After growth for 24 hours, the fermentate extracts (cell free supernatants) were obtained by centrifugation and filter-sterilization of the fermentate.

[0419] Normal human epidermal keratinocytes (NHEK) isolated from a single adult donor were thawed in Keratinocyte Growth Media 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 pg / mL), hydrocortisone (0.33 pg / mL), epinephrine (0.39 pg / mL), transferrin (10 pg / mL) and calcium chloride (0.06 mM). NHEK were subsequently cultured in a T75 flask containing KGM2 to achieve 75-80% confluency in a 37°C incubator supplied with 5% CO2 and 95% humidity. Following reaching desired confluency, NHEK were removed from the T75 flask using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK were resuspended in KGM2, counted using a hemocytometer and seeded onto transwell inserts (6.5 mm insert; 0.4 pm polyester membrane; tissue culture treated, polystyrene 24 well plate). NHEK were incubated in a 37°C incubator supplied with 5% CO2 and 95% humidity until cells reached 100% confluency on the transwell membrane. At that point, the media was aspirated and NHEK were subsequently grown in KGM2 supplemented with 1 .5 mM calcium chloride instead of 0.06 mM for remainder of experiment. To the top well, B. subtilis fermentate extract was added such that the final fermentate extract concentration was 0.1 % by volume. Following addition of fermentate extract, the transepidermal electrical resistance (TEER) was measured every day using an epithelial volt / ohm meter (EVOM). Media and fermentate extract were refreshed every 2 days. Results are shown in Table 9 and compared to the untreated (no fermentate extract) control.

[0420] Table 9. B. subtilis fermentate extract (cell free supernatant) of sporulation spollE knockout further increases Transepidermal Electrical Resistance TEER

[0421] The results in Table 7 indicate that addition of 0.1 % of B. subtilis G01_spo fermentate extract (cell free supernatant) has a higher percent increase in TEER value when compared to the addition of 0.1 % of B. subtilis G01 , which is an indication of further improved skin barrier function, compared to an untreated (no fermentate extract)control sample.

[0422] EXAMPLE 13

[0423] Knocking out B. subtilis sporulation gene spo / / E further enhances the ability of B. subtilis fermentate extracts to inhibit growth of Malassezia

[0424] The ability of B. subtilis G01_spo fermentate extract to inhibit the growth of Malassezia species was evaluated. The B. subtilis G01_spo variant strain (CBS151098) and its parent strain B. subtilis G01 (CBS147469) were grown in cultivation medium. The cultivation medium used urea as the main nitrogen source and glucose as the main carbon source and was supplemented with 1 % soytone. Generation of the fermentate was carried out in 50-mL centrifuge tubes with 5 mL medium. The flasks were placed in a platform shaker with a constant speed of 200 rpm at 32°C for 24 h. The generation of the fermentate extract (cell free supernatant) was obtained by spinning down the fermentate in a centrifuge at 4000 x g for 10 minutes, then the cell free supernatant was filtered using a 0.22 pm low- protein binding polyethersulfone (PES) membrane filter. The resulting fermentate extract (cell free superatant) was stored at 4°C for later use.

[0425] Malassezia furfur strain DSM 6170 was grown in modified Dixon (mDixon) broth. Each liter of mDixon broth contained 36 g malt extract, 20 g desiccated oxbile, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. Generation of the culture was carried out in a 125-mL flat-bottom shake flask with 25 mL of medium. The flask was placed in a platform shaker with a constant speed of 200 rpm at 32°C for 48 h. The culture was then diluted in 1 % Tween-40 to an ODeoo of 0.42 (1 .0 x 107CFU / mL). A volume of 100 pL of the diluted culture was spread onto mDixon agar plates and allowed to dry. Each liter of mDixon agar contained 36 g malt extract, 20 g desiccated ox-bile, 15 g Bacto agar, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. The B. subtilis G01 and B. subtilis G01_spo fermentate extracts were spotted onto the center of the M. furfur plates in 2.5 pL aliquots. The plates were incubated at 32°C overnight.

[0426] Following the overnight incubation, the plates were analyzed for the sizes of the growth inhibition zones created by the fermentate extracts. The assay was run in triplicates and average values are shown in Table 10.

[0427] Table 10: Growth inhibition assay of Malassezia using zone inhibition assay with fermentate extracts of B. subtilis G01 and B. subtilis G01 spo fermentate extracts The results in Table 10 show that the B. subtilis G01_spo fermentate extract (cell free supernatant) has a larger growth inhibition zone when compared to B. subtilis G01 parental strain, which is an indication that the fermentate extract obatined from a B. subtilis spollE knockout variant strain further improves Malassezia growth inhibition activity.

[0428] In order to evaluate the specificity of the activity against other skin microbes by fermentate extracts of B. subtilis G01_spo, Staphylococcus hominis ATCC 27844 Staphylococcus epidermidis ATCC 12228 and Cutibacteria acnes K2 strains were used as target organisms in a zone inhibition assay. Fermentate for B. subtlis G01_spo strain was generated as described in Example 4. The strain was grown in SUM (Soyptone Urea Micronutrient) medium. Each liter of SUM medium (1 .0 X strength) consists of 10 g soytone, 75 g glucose, 3 ml of dipotassium phosphate (1 M), 3.6 g urea, and 10 ml of micronutrient stock. Each liter of the micronutrient stock contained 1.47g Sodium Citrate 2H2O, 1.47 CaCI2-2 H2O, 0.4 FeSO4 -7 H2O, 0.1 g MnSO4- H2O, 0.1 g ZnSO4- H2O, 0.05 g CuCI2.2 H2O, 0.1 g CoCI2 6 H2O, and 0.1 g Na2MoO4 2 H2O. The SUM medium was adjusted to pH 7.3. The SUM medium at 0.5 x strength was obtained by diluting the SUM medium by half with purified water. Generation of B. subtilis fermentate was carried out in 125 ml flat bottom shake flasks with 25 ml SUM medium at 0.5x strength. The flasks were placed in a platform shaker with a constant speed of 200 rpm at 32oC. The overnight fresh seed culture was used as inoculum and the starting OD was 0.125 at 600 nm. After growth for 24 hours, the cell free supernatant (fermentate extract) was obtained by centrifugation and filtersterilization.

[0429] Direct spotting of the 5 pl of the fermentate extract of B. subtilis G01_spo did not result in visible clearing zones for both organisms in the zone inhibition assay against Malassezia. This result indicated that the fermentate extract generated from B. subtilis G01_spo was similar to the parent strain of B. subtilis G01 strain in this regard. Both fermentate extract samples had high specificity for growth inhibition of Malassezia without affecting growth of Staphylococcus hominis, Staphylococcus epidermidis and Cutibacteria acnes strains. In order to elucidate the range of molecular weight of the bioactive(s) associated with the growth inhibition of Malassezia by the B. subtilis G01-spo strain, the fermentate extract was filtered through a membrane with a 3 KDa cut off. The flow through was collected and used in the zone inhibition assay against Malassezia furfur strain DSM 6170. The size of the zone was 53 mm for the fermentate extract before filtering and the size of the zone for the flow through was 50 mm. These results indicate that the less than 3 KDa flow through of the Bacillus subtilis fermentate extract contains acitve agent(s) of less than 3 kDa capable of inhibiting the growth inhibition activity against of Malassezia.

[0430] EXAMPLE 14

[0431] Knocking out B. subtilis surfactin operon s / f does not decrease the ability of B. subtilis fermentate extracts to inhibit Malassezia growth

[0432] B. subtilis strains produce various biologically active metabolites that have the ability to inhibit the growth of other microorganisms such as fungi and bacteria. Among these metabolites, surfactin (sr SEQ ID NO: 16) is a potent lipopeptide- based biosurfactant (Cheng et al., 2023, AIMS Microbiology, 2023, Vol 9 (2), 195- 217).

[0433] The ability for the surfactin produced by B. subtilis G01 (CBS1 7469) to inhibit Malassezia was evaluated.

[0434] A CRISPR-Cas9 plasmid (referred to as pMY1 -6.2) was constructed to delete the srf locus from B. subtilis G01 (CBS147469). The pMY1 -6.2 plasmid comprised a Streptococcus pyogenes Cas9 endonuclease expression cassette as well as an editing template insert (SEQ ID NO: 17), containing the B. subtilis G01 homologous regions and the sgRNA (SEQ ID NO: 18), and the kanamycin resistance cassette. The pMY1-6.2 plasmid was electroporated into Endura™ E I ectrocom petent Cells (Biosearch Technologies). The assembled construct was verified via Sanger seguencing. Rolling-circle amplification (RCA) was used to amplify pMY1 -6.2 using the GenomiPhi HY Ready-To-Go DNA Amplification Kit (Cytiva). The B. subtilis G01 (CBS147469) was modified to contain the competence plasmid pBL.comK, as described in WO2019 / 40423, and a deletion of the srf operon. Briefly, pBL.comK was transformed into B. subtilis G01 using natural competence and with spectinomycin as the resistance marker. Then, the pBL.comK-containing strain was made competent, as described in WO2021 / 146411 , and subsequently transformed with plasmid pMY1 -6.2. A volume of 200 pL competent cells was mixed with 50 pL pMY1 -6.2 RCA product and 1 mL Luria-Bertani (LB) broth and then 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 for cells transformed with the Cas9 plasmid pMY1 -6.2. The isolates were screened for the deletion of the s / Toperon using Q5® PCR with primer BSsrf-checkdel-F (SEQ ID NO: 19) and primer BSsrf-checkdel-R (SEQ ID NO: 20). Colonies with the srf deletion (As / f) produced a PCR product of 2604 bp, whereas the wild-type strain containing the intact srf operon produced a PCR product too large to view on the agarose gel (28,581 bp). Deletions were confirmed via Sanger sequencing. To further confirm that the knockout isolates were not mixed populations of the srf knockout and the wild-type strain, a multiplex PCR was carried out to confirm that the individual genes in the srf operon were deleted. If the colonies were mixed populations, 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 a gene did not produce a band for that gene. Confirmed mutant isolates were stored as glycerol stocks at -80°C.

[0435] The isolates were cured of pBL.comK and pMY1 -6.2 by culturing them in tryptic soy broth (TSB) containing no antibiotics for three days, passaging once every 24 hours, and then plating onto plain tryptic soy agar (TSA). Colonies were selected from the TSA and patched onto LB plates containing no antibiotics, LB containing 20 ppm kanamycin, and LB plates containing 100 ppm spectinomycin. Cured isolates grew on plain LB and not LB containing kanamycin or spectinomycin.

[0436] Following genomic confirmation of the srf deletion, phenotypic analysis of the B. subtilis G01 lacking the srf locus, referred to as B. subtilis G01_s / f , using liquid chromatography-ultraviolet (LC-UV) was conducted on the fermentate extracts (cell free supernatant) to confirm the loss of surfactin production in B. subtilis G01_srf. The B. subtilis G01_s / f variant strain and its parent strain B. subtilis G01 (CBS147469) were grown in cultivation medium. The cultivation medium used urea as the main nitrogen source and glucose as the main carbon source and was supplemented with 1 % soytone. Generation of the fermentate was carried out in 50-mL centrifuge tubes with 5 mL medium. The flasks were placed in a platform shaker with a constant speed of 200 rpm at 32°C for 24 h. The generation of the fermentate extract (cell-free supernatant) was obtained by spinning down the fermentate in a centrifuge at 4000 x g for 10 minutes, then the cell free supernatant was filtered using a 0.22 pm low-protein binding polyethersulfone (PES) membrane filter.

[0437] The resulting fermentate extracts were analyzed by LC-UV, and the samples were quantitated using a calibration curve prepared with surfactin standard. The results are shown in Table 11 .

[0438] Table 11 : B. subtilis G01_s / f does not produce surfactin.

[0439] The results presented in Table 11 show that the B. subtilis G01 wild type produced 318.1 mg / L of surfactin, whereas the B. subtilis G01 As / f isolates had no surfactin detectedTherefore, the deletion of the srf operon removed B. subtilis G01 ’s ability to produce surfactin.

[0440] Following phenotypic confirmation of the surfactin knockout (8. subtilis G0'\_srf), the ability of B. subtilis G01 _srf fermentate extract to inhibit the growth of Malassezia species was evaluated. The B. subtilis G01 _srf variant strain and its parent strain B. subtilis G01 (CBS147469) were grown in cultivation medium as previously described. The resulting fermentate extracts (cell-free supernatant) were stored at 4°C for later use.

[0441] Malassezia furfur strain DSM 6170 was grown in modified Dixon (mDixon) broth. Each liter of mDixon broth contained 36 g malt extract, 20 g desiccated oxbile, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. Generation of the culture was carried out in a 125-mL flat-bottom shake flask with 25 mL of medium. The flask was placed in a platform shaker with a constant speed of 200 rpm at 32°C for 48 h. The culture was then diluted in 1 % Tween-40 to an ODeoo of 0.42 (1 .0 x 107CFU / mL). A volume of 100 pL of the diluted culture was spread onto mDixon agar plates and allowed to dry. Each liter of mDixon agar contained 36 g malt extract, 20 g desiccated ox-bile, 15 g Bacto agar, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. The B. subtilis G01 wild-type and B. subtilis G01 As / f fermentate extracts were spotted onto the center of the M. furfur plates in 2.5 pL aliquots. The plates were incubated at 32°C overnight.

[0442] Following the overnight incubation, the plates were analyzed for the sizes of the growth inhibition zones created by the fermentate extracts. The assay was run in triplicates and average values are shown in Table 12.

[0443] Table 12: Growth inhibition assay of Malassezia using zone inhibition assay with fermentate extracts (cell-free supernatant) of B. subtilis G01 and B. subtilis G01 As / f.

[0444] The results presented in Table 12 show that the B. subtilis GO1_srf fermentate extract (cell free supernatant) has no reduced inhibition zone when compared to B. subtilis G01 parental strain, which is an indication that the surfactin produced by B. subtilis G01 does not contribute to Malassezia growth inhibition activity.

[0445] EXAMPLE 15

[0446] Improvement of skin barrier function using a less than 3k Da B. subtilis fermentate extract fraction.

[0447] The ability of the less than 3 kilodalton (kDa) fraction of B. subtilis fermentate extract to improve the skin barrier function was evaluated. B. subtilis G01_spo strain was grown in modified SMU media consisting of 1 g / L soytone, 3mM KH buffer, 20 g / L glucose, 1 .8 g / L urea, 0.735 g / L Sodium Citrate 2H2O, 0.735 g / L CaCI2 2 H2O, 0.2 g / L FeSO4 7 H2O, 0.05 g / L MnSO4 H2O, 0.05 g / L ZnSO4- H2O, 0.0025 g / L CuCI2.2 H2O, 0.05 g / L CoCI2-6 H2O, and 0.05 g / L Na2MoO4 2 H2O. After growth for 24 hours, the fermentate extract (cell free supernatant) was obtained by centrifugation and filter-sterilization of the fermentate. Following centrifugation and filter sterilization, the fermentate extract was then fractionated by molecular weight by passing the fermentate extract through a series of molecular weight cut-off filters in the following progression: 30 kilodalton (kDa), 10 kDa, and 3 kDa. Fermentate extracts that did not pass through the filters were saved and referred to as retentates (R) and fermentate extract that passed through the filters were saved and referred to as flow throughs (FT). A portion of the 3 kDa FT was subsequently concentrated by a factor of 10 using a speedvac. Briefly, 3 mL of the 3 kDa FT fraction was centrifuged under vacuum at room temperature until the final volume was 300 pL. This fraction will be referred to as 3 kDa FT 10X. The 3 kDa FT unconcentrated and 3 kDa FT 10x fractions of B. subtilis G01_spo strain were tested for the ability to improve skin barrier function using NHEK as performed in Example 12 and results are compared against a untreated (no fermentate extract) control. Results are shown in Table 13.

[0448] Table 13. The < 3 kPa flow through B. subtilis molecular weight fermentate extract fraction increases Transepidermal Electrical Resistance (TEER).

[0449] The results in Table 13 indicate that addition of the less than 3 kDa unconcentrated and concentrated forms of B. subtilis G01_spo fermentate extract (cell free supernatant) has a higher percent increase in TEER value when compared to the untreated (no fermentate extract) control , which is an indication of improved skin barrier function.

[0450] EXAMPLE 16

[0451] Knocking out B. subtilis bacilysin operon bac decreases the ability of B. subtilis fermentate extracts to inhibit Malassezia growth

[0452] The ability for the B. subtilis bacilysin operon bac (bac A-bac B-bac C-bac D-bac E-bac F-bac G, SEQ ID NO:21 ) to produce a bacilysin type dipeptide to inhibit Malassezia was evaluated. A CRISPR-Cas9 plasmid (referred to as pMY1 -13.1 ) was constructed to delete the bac locus from B. subtilis G01 (CBS147469). The pMY1 -13.1 plasmid comprised a Streptococcus pyogenes Cas9 endonuclease expression cassette as well as an editing template insert (SEQ ID NO: 22), containing the B. subtilis G01 homologous regions and the sgRNA (SEQ ID NO: 23), and the kanamycin resistance cassette. The pMY1-13.1 plasmid was electroporated into Endura™ E I ectrocom petent Cells (Biosearch Technologies). The assembled construct was verified via Sanger sequencing. Rolling-circle amplification (RCA) was used to amplify pMY1 -13.1 using the GenomiPhi HY Ready-To-Go DNA Amplification Kit (Cytiva).

[0453] The B. subtilis G01 (CBS147469) was modified to contain the competence plasmid pBL.comK, as described in WO2019 / 40423, and a deletion of the bac operon. Briefly, pBL.comK was transformed into B. subtilis G01 using natural competence and with spectinomycin as the resistance marker. Then, the pBL.comK-containing strain was made competent, as described in WO2021 / 146411 , and subsequently transformed with plasmid pMY1 -13.1 . A volume of 200 pL competent cells was mixed with 50 pL pMY1-13.1 RCA product and 1 mL Luria-Bertani (LB) broth and then 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 for cells transformed with the Cas9 plasmid pMY1 -13.1 . The isolates were screened for the deletion of the bac operon using Q5® PCR with primer BSbac- checkdel-F (SEQ ID NO: 24) and primer BSbac-checkdel-R (SEQ ID NO: 25). Colonies with the bac deletion (Abac) produced a PCR product of 2417 bp, whereas the wild-type strain containing the intact bac operon produced a PCR product of 9121 bp. Deletions were confirmed via Sanger sequencing. Confirmed mutant isolates were stored as glycerol stocks at -80°C.

[0454] The isolates were cured of pBL.comK and pMY1 -13.1 by culturing them in tryptic soy broth (TSB) containing no antibiotics for three days, passaging once every 24 hours, and then plating onto plain tryptic soy agar (TSA). Colonies were selected from the TSA and patched onto LB plates containing no antibiotics, LB containing 20 ppm kanamycin, and LB plates containing 100 ppm spectinomycin. Cured isolates grew on plain LB and not LB containing kanamycin or spectinomycin.

[0455] Following genotypic confirmation of the bac operon knockout, the ability of the B. subtilis G01 bacilysin knockout mutants, henceforth referred to as B. subtilis G01 _bac, to inhibit the growth of Malassezia species was evaluated. The B. subtilis G01_bac variant strain and its parent strain B. subtilis G01 (CBS147469) were grown in cultivation medium. The cultivation medium used urea as the main nitrogen source and glucose as the main carbon source and was supplemented with 1 % soytone. Generation of the fermentate was carried out in 50-mL centrifuge tubes with 5 mL medium. The flasks were placed in a platform shaker with a constant speed of 200 rpm at 32°C for 24 h. The generation of the fermentate extract (cell-free supernatant) was obtained by spinning down the fermentate in a centrifuge at 4000 x g for 10 minutes, then the cell-free supernatant was filtered using a 0.22 pm low-protein binding polyethersulfone (PES) membrane filter. The resulting fermentate extracts (cell-free supernatant) were stored at 4°C for later use.

[0456] Malassezia furfur strain DSM 6170 was grown in modified Dixon (mDixon) broth. Each liter of mDixon broth contained 36 g malt extract, 20 g desiccated oxbile, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. Generation of the culture was carried out in a 125-mL flat-bottom shake flask with 25 mL of medium. The flask was placed in a platform shaker with a constant speed of 200 rpm at 32°C for 48 h. The culture was then diluted in 1 % Tween-40 to an ODeoo of 0.42 (1 .0 x 107CFU / mL). A volume of 100 pL of the diluted culture was spread onto mDixon agar plates and allowed to dry. Each liter of mDixon agar contained 36 g malt extract, 20 g desiccated ox-bile, 15 g Bacto agar, 6 g peptone, 2 mL glycerol, 2 mL oleic acid, and 10 mL Tween-40. The B. subtilis G01 wild-type and B. subtilis G01 bac fermentate extracts were spotted onto the center of the M. furfur plates in 2.5 pL aliquots. The plates were incubated at 32°C overnight.

[0457] Following the overnight incubation, the plates were analyzed for the sizes of the growth inhibition zones created by the fermentate extracts. The assay was run in triplicates and average values are shown in Table 14. Table 14: Growth inhibition assay of Malassezia using zone inhibition assay with fermentate extracts (cell-free supernatant) of B. subtilis G01 and B. subtilis G01 bac. The results presented in Table 14 show that the B. subtilis G01_bac fermentate extract (cell free supernatant) produces no zone of inhibition, whereas the B. subtilis G01 parental strain produces a zone of 36 mm. This demonstrates that the deletion of the bac operon removes the ability for B. subtilis G01 to inhibit the growth of Malassezia and, therefore, indicates that this pathway contributes to the ability of B. subtilis G01 to inhibit Malassezia growth.

Claims

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

2. The skin care composition of claim 1 , wherein the Bacillus subtilis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus subtilis fermentate, a cell pellet extract obtained from a Bacillus subtilis fermentate, a whole broth fermentate extract obtained from a Bacillus subtilis fermentate, and any one combination thereof.

3. The skin care composition of claim 1 , wherein the scalp or skin disorder is selected from the group consisting of an unbalanced ecoflora of the scalp, discomfort of the scalp, tinea versicolor, dry skin, irritated skin, and any one combination thereof.

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

5. The skin care composition of any preceding claim, wherein the Bacillus subtilis fermentate extract, or fraction thereof, is obtained from a fermentate of a Bacillus subtilis selected from the group consisting of a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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 to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of 8. subtilis G01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) undernumber CBS149857, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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 to a 16S ribosomal RNA sequence (SEQ ID NO: 2) of Bacillus subtilis PC01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149969, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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 to a 16S ribosomal RNA sequence (SEQ ID NO: 3) of Bacillus subtilis N01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149971 , a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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 to a 16S ribosomal RNA sequence (SEQ ID NO: 7) of Bacillus subtilis K01 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS149972, a B. subtilis strain having a 16S ribosomal RNA sequence displaying 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 to a 16S ribosomal RNA sequence (SEQ ID NO: 1 ) of B. subtilis G01_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151098, and any one combination thereof.

6. The skin care composition of any preceding claim, further comprising one or more antidandruff active agents.

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

8. The skin care composition of claim 1 , wherein said skin barrier strengthening and / or said improving skin barrier function and / or said skin moisturization occursthrough fatty acid synthesis, cornification, small proline rich proteins synthesis, or any one combination thereof.

9. The skin care composition of any preceding claim, further comprising an additional compound selected from the group consisting of a fragrance, an excipient, a preservative, a pH adjuster, an aqueous carrier, an alcohol carrier, an emollient, a solidification agent, a hydration agent, an emulsifier, a solubilizer, a surfactant, a thickening agent, a salt and any one combination thereof.

10. Use of the skin care composition of any preceding claim in a cosmetic skin care product.11 . A skin care product comprising the skin care composition of any preceding claim and one or more dermatologically or skin care acceptable component.

12. The skin care product of claim 11 , wherein said 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% up to 10% of said skin care composition on a weight basis relative to a total weight of said skin care product.

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

14. The skin care product of any one of claim 11 -13, wherein the skin care product is selected from the group consisting of a lotion, a serum, a jelly, a cream, a gel, a hydrogel, an emulsion, a solid cosmetic, a mask, a patch, a shampoo, a loose or compact powder, a liquid suspension or solution, a spray solution and a stick.

15. A method for providing at least one skin care benefit to a subject in need thereof, the method comprising topically administering a skin care composition or skin care product of any preceding claim to the subject’s skin or scalp.

16. A method for reducing dandruff in a subject, the method comprising topically administering a skin care composition or skin care product of any preceding claim to the scalp of said subject.

17. A method for skin barrier strengthening in a subject, the method comprising topically administering a skin care composition or skin care product of any preceding claim, to the skin or scalp of said subject.

18. A method for skin moisturization in a subject, the method comprising topically administering a skin care composition or skin care product of any preceding claim to the skin or scalp of said subject.

19. The method of any one of claim 15 - claim 18, wherein the Bacillus subtilis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus subtilis fermentate, a cell pellet extract obtained from a Bacillus subtilis fermentate, a whole broth fermentate extract obtained from a Bacillus subtilis fermentate, and any one combination thereof.

20. A Bacillus subtilis strain variant fermentate, Bacillus subtilis strain variant fermentate extract, or fraction of said variant fermentate, of a B. subtilis strain variant, wherein said Bacillus subtilis strain variant is derived from a parental B. subtilis by knocking out at least one sporulation gene of said parental B. subtilis strain, wherein said variant fermentate, said variant fermentate extract, or said fraction of said variant fermentate, has increased activity when compared to the activity of a fermentate, a fermentate extract, or fraction thereof derived from said parental B. subtilis strain, wherein said activity is selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.21 . A method for increasing the activity of a Bacillus subtilis strain fermentate extract for providing at least one skin care benefit, the method comprising:(a) providing a parent Bacillus subtills strain capable of producing an effective amount of a fermentate extract for providing at least one skin care benefit selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof;(b) modifying said parent Bacillus subtilis strain by knocking out at least one sporulation gene from the genome of said Bacillus subtilis strain, thereby producing a variant strain that is unable to form spores; and,(c) producing a fermentate extract of said Bacillus subtilis variant strain, wherein said fermentate extract of said Bacillus subtilis variant strain has an increased activity for providing at least one skin care benefit selected from the group consisting of reducing dandruff, reducing a scalp or skin disorder, skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof, when compared to the activity of the fermentate extract of said parent Bacillus subtilis strain.

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