Fermentate extracts for providing skin care benefits and use 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 velezensis fermentate, a Bacillus velezensis fermentate extract, or fraction thereof, for providing at least one skin care benefit in a subject in need thereof, including skin barrier strengthening, improving skin barrier function, skin moisturization and promoting skin repair.

Description

[0001] TITLE

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

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 567133 filed March 19, 2024, which is incorporated by reference herein in its 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 velezensis fermentate, a Bacillus velezensis fermentate extract, or fraction thereof, for providing at least one skin care benefit in a subject in need thereof, including skin barrier strengthening, improving skin barrier function, skin moisturization and promoting skin repair.

[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_NB42137PCT_SequenceListing. xml; Size: 12,460 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).

[0010] 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.

[0011] 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).

[0012] 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).

[0013] 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).

[0014] Keratinocytes in the stratum corneum, 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 cross- link 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)

[0015] 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, bums 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 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.

[0016] 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).

[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] 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.

[0020] SUMMARY

[0021] 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 velezensis fermentate, Bacillus velezensis fermentate extract, or fraction thereof, for providing at least one skin care benefit in a subject in need thereof, including skin barrier strengthening, improving skin barrier function, skin moisturization and promoting skin repair. Furthermore, methods for increasing the activity of a Bacillus velezensis strain fermentate extract for providing at least one skin care benefit are disclosed herein.

[0022] The inventors have unexpectedly observed that a fermentate extract (cell free supernatant) of Bacillus velezensis (such as but not limited to B. velezensis E04 and B. velezensis E04_spo) strengthens the skin barrier and improves the skin barrier function as well as promotes skin repair.

[0023] 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 velezensis 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, promoting skin repair, and any one combination thereof.

[0024] In one embodiment, the skin care composition is a composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus velezensis 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, promoting skin repair, and any one combination thereof, wherein the Bacillus velezensis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus velezensis fermentate, a whole broth fermentate extract obtained from a Bacillus velezensis fermentate, and any one combination thereof.

[0025] In one embodiment, the skin care composition is a skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus velezensis 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, promoting skin repair, and any one combination thereof, wherein said skin barrier strengthening and / or said improving skin barrier function and / or said skin moisturization, occurs through increased tight junctions, increased desmosomes, increased cell-cell adhesion, increased cornification, and any one combination thereof.

[0026] In one aspect, the promoting skin repair occurs through proliferation and migration of keratinocytes.

[0027] In one aspect the skin care composition described herein 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.

[0028] In one embodiment, the composition is a Bacillus velezensis strain variant fermentate, Bacillus velezensis strain variant fermentate extract, or fraction of said variant fermentate, of a B. velezensis strain variant, wherein said Bacillus velezensis strain variant is derived from a parental B. velezensis by knocking out a sporulation gene of said parental B. velezensis 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. velezensis strain, wherein said activity is selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0029] In one embodiment, the skin care product is a skin care product comprising a skin care composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus velezensis 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, promoting skin repair, and any one combination thereof.

[0030] 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 of said subject, wherein said composition comprises an effective amount of a B. velezensis 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, promoting skin repair, and any one combination thereof.

[0031] In one embodiment, the method is a method for increasing the activity of a Bacillus velezensis strain fermentate extract for providing at least one skin care benefit selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof.

[0032] DETAILED DESCRIPTION

[0033] 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.

[0034] Microorganisms, Fermentates, and Fermentate Extracts

[0035] As used herein, "microorganism" or “microbe” refers to a bacterium, a fungus, a virus, a protozoan, archaea, and other microbes or microscopic organisms.

[0036] In some embodiments, the Bacillus velezensis (B. velezensis) cells suitable for use in the present invention can be subjected to treatments that render them non-replicating, for example, exposure to heat, desiccation, y-irrad iation, or UV- irradiation. A non-replicating 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 non-replicating 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.

[0037] 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.

[0038] In one aspect, the microorganism suitable for use in the present invention includes strains of Bacillus velezensis.

[0039] In some embodiments, the microorganism suitable for use in the present invention includes 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 of B. velezensis E04 (SEQ ID NO: 1 ) deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469.

[0040] The 16S ribosomal RNA sequence of Bacillus velezensis E04 is as follows:

[0041] CGAUGCGUAGCCGACCUGAGAGGGUGAUCGGCCACACUGGGACUGA

[0042] GACACGGCCCAGACUCCUACGGGAGGCAGCAGUAGGGAAUCUUCCGCAAU GGACGAAAGUCUGACGGAGCAACGCCGCGUGAGUGAUGAAGGUUUUCGGA UCGUAAAGCUCUGUUGUUAGGGAAGAACAAGUGCCGUUCAAAUAGGGCGG CACCUUGACGGUACCUAACCAGAAAGCCACGGCUAACUACGUGCCAGCAGC CGCGGUAAUACGUAGGUGGCAAGCGUUGUCCGGAAUUAUUGGGCGUAAAG GGCUCGCAGGCGGUUUCUUAAGUCUGAUGUGAAAGCCCCCGGCUCAACCG GGGAGGGUCAUUGGAAACUGGGGAACUUGAGUGCAGAAGAGGAGAGUGGA AUUCCACGUGUAGCGGUGAAAUGCGUAGAGAUGUGGAGGAACACCAGUGG CGAAGGCGACUCUCUGGUCUGUAACUGACGCUGAGGAGCGAAAGCGUGGG GAGCGAACAGGAUUAGAUACCCUGGUAGUCCACGCCGUAAACGAUGAGUGC UAAGUGUUAGGGGGUUUCCGCCCCUUAGUGCUGCAGCUAACGCAUUAAGC ACUCCGCCUGGGGAGUACGGUCGCAAGACUGAAACUCAAAGGAAUUGACG GGGGCCCGCACAAGCGGUGGAGCAUGUGGUUUAAUUCGAAGCAACGCGAA GAACCUUACCAGGUCUUGACAUCCUCUGACAAUCCUAGAGAUAGGACGUCC CCUUCGGGGGCAGAGUGACAGGUGGUGCAUGGUUGUCGUCAGCUCGUGU CGUGAGAUGUUGGGUUAAGUCCCGCAACGAGCGCAACCCUUGAUCUUAGU UGCCAGCAUUCAGUUGGGCACUCUAAGGUGACUGCCGGUGACAAACCGGA GGAAGGUGGGGAUGACGUCAAAUCAUCAUGCCCCUUAUGACCUGGGCUAC ACACGUGCUACAAUGGACAGAACAAAGGGCAGCGAAACCGCGAGGUUAAGC CAAUCCCACAAAUCUGUUCUCAGUUCGGAUCGCAGUCUGCAACUCGACUGC GUGAAGCUGGAAUCGCUAGUAAUCGCGGAUCAGCAUGCCGCGGUGAAUAC GUUCCCGGGCCUUGUACACACCGCCCGUCACACCACGAGAGUUUGUAACAC CCGAAGUCGGUGAGGUAACCUUUUAGGAGCCAGCCGCCGAAGGUGGGACA GAUGAUUGGGGUGAAGUCGUAACAAGGUAGCCGUAUCGGAAGGUGCGGCU

[0043] GGAUCACCUCCUUU (SEQ ID NO: 1 ).

[0044] In some embodiments, the microorganism suitable for use in the present invention includes 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 of B. velezensis E04 (SEQ ID NO: 1 ), and wherein the sporulation gene spollE has been knocked out, referred to as B. velezensis E04_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099.

[0045] In one aspect of the invention, fermentates are provided.

[0046] 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).

[0047] Fermentates for use in the present invention include fermentates from the microorganism Bacillus velezensis.

[0048] 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.

[0049] In one aspect, the fermentates for use in the present invention includes fermentates from the microorganism Bacillus velezensis, wherein said B. velezensis is selected from a Bacillus velezensis 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 of B. velezensis E04 (SEQ ID NO: 1 ) deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469.

[0050] In one aspect, the fermentates for use in the present invention includes fermentates from the microorganism Bacillus velezensis, wherein said B. velezensis is selected from a Bacillus velezensis 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 of B. velezensis E04_spo (SEQ ID NO: 1 ) deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099X.

[0051] Fermentates can be further concentrated prior to be 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 can be spray-dried or lyophilized prior to be include in a composition.

[0052] 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.

[0053] 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 velezensis.

[0054] 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 velezensis species. 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 velezensis. 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 velezensis cells were cultivated.

[0055] In one aspect, the cell free supernatant (also referred to as fermentate extract) is obtained by filtration or centrifugation of a Bacillus velezensis fermentate.

[0056] In one aspect, cells were removed from the Bacillus velezensis 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.

[0057] In one aspect, the fermentate extract for use in the present invention is a Bacillus velezensis 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. The fermentate extract for use in the compositions and methods and / or uses of the present invention may be substantially free of viable Bacillus velezensis cells, typically containing zero (or substantially zero) viable cells / mL fermentate.

[0058] 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.

[0059] In one aspect, the cell pellet extract is obtained from a Bacillus velezensis 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. 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.

[0060] In another aspect, Bacillus velezensis fermentate extracts are produced by combining the cell free supernatant fraction described above with the cell pellet extract described above.

[0061] In yet another aspect, the Bacillus velezensis 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 (also referred to as “whole broth fermentate extract”. 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 composition.

[0062] Alternatively, the Bacillus velezensis fermentate extract is a whole broth 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”).

[0063] In one aspect the fermentate extract is obtained from a fermentate that was produced with a nutrient medium having a pH between 2-12.

[0064] 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.

[0065] 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 velezensis fermentate extracts can be observed.

[0066] In one embodiment, the Bacillus velezensis fermentate, fermentate extract, or composition comprising the fermentate extract is formulated in a dry formulation or a liquid formulation.

[0067] In one embodiment, the Bacillus velezensis 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.

[0068] In one aspect the granule comprises the Bacillus velezensis 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.

[0069] 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 velezensis 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

[0070] In one aspect, the Bacillus velezensis fermentate, Bacillus velezensis fermentate extract, or fraction thereof, has efficacy in providing at least one skin care benefit selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturizing, promoting skin repair, and any one combination thereof.

[0071] 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.

[0072] 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 velezensis, wherein said Bacillus velezensis is selected from a Bacillus velezensis 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 of B. velezensis E04 (SEQ ID NO: 1 ) deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469.

[0073] In one aspect, the fermentate extracts, or fractions thereof, for use in the present invention includes fermentate extracts from the microorganism Bacillus velezensis, wherein said B. velezensis is selected from a B. velezensis 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 of B. velezensis E04_spo (SEQ ID NO: 1 ) deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099, wherein the sporulation gene spollE has been knocked out.

[0074] Skin care compositions and skin care products for providing at least one skin care benefit.

[0075] 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.

[0076] As used herein the term “skin care benefit agent” or “active agent” or “bioactive” are used interchangeably, and refer to a Bacillus velezensis fermentate, Bacillus velezensis fermentate extract, or fraction thereof, that can provide a skin care (scalp care) benefit.

[0077] In some embodiments provided herein, the skin care benefit agent includes an effective amount of a Bacillus velezensis fermentate, Bacillus velezensis fermentate extract, or fraction thereof, wherein said Bacillus velezensis is selected from a B. velezensis 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 of B. velezensis E04 (SEQ ID NO: 1 ) deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, and / or a B. velezensis 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 of B. velezensis E04_spo (SEQ ID NO: 1 ) deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099.

[0078] 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 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 scalp. Unexpectedly, the inventors have observed that a fermentate extract (cell free fermentate supernatant) of Bacillus velezensis strengthens the skin barrier (improves the keratinocyte barrier) and improves the skin barrier function.

[0079] In one aspect of the invention the skin care benefit is selected from the group consisting of 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.

[0080] In one aspect the skin care composition for providing at least one skin care benefit in a subject in need thereof comprises an effective amount of a Bacillus velezensis fermentate extract, wherein said effective amount of said Bacillus velezensis fermentate extract is 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%,

[0081] 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%,

[0082] 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%,

[0083] 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%,

[0084] 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%,

[0085] 99% up to 100 % on a weight basis relative to a total weight of said skin care composition.

[0086] In one aspect the skin care composition for providing at least one skin care benefit in a subject in need thereof comprises an effective amount of a Bacillus velezensis fermentate extract, wherein said effective amount of said Bacillus velezensis fermentate extract is at least about 0.01 %, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 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 % on a volume basis relative to the total volume of said skin care composition.

[0087] 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 velezensis 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, promoting skin repair, and any one combination thereof.

[0088] 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 velezensis 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, promoting skin repair, and any one combination thereof, wherein said skin barrier strengthening and / or improving skin barrier function and / or skin moisturization and / or promoting skin repair comprises administering the composition to the subject’s skin or scalp to strengthen the skin barrier of said subject and / or improve the skin barrier function and / to moisturize the skin and / or to promote skin repair.

[0089] In one aspect the composition promotes skin repair through production of flagellin.

[0090] 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 velezensis 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, promoting skin repair, and any one combination thereof, wherein said composition comprises an effective amount of a Bacillus velezensis 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%, 35.0%, 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 100% by weight relative to a total weight of said composition.

[0091] 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 velezensis 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, promoting skin repair, and any one combination thereof, wherein said composition comprises an effective amount of a Bacillus velezensis 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%, 35.0%, 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 volume relative to a total volume of said composition.

[0092] In one embodiment, the skin care composition is a composition for providing at least one skin care benefit in a subject in need thereof, the composition comprising an effective amount of a Bacillus velezensis 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, promoting skin repair, and any one combination thereof, wherein the Bacillus velezensis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus velezensis fermentate, a whole broth fermentate extract obtained from a Bacillus velezensis fermentate, and any one combination thereof.

[0093] In one embodiment, the skin care composition is a composition for providing at least one skin care benefit described herein, wherein the Bacillus velezensis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus velezensis fermentate, a cell pellet extract obtained from a Bacillus velezensis fermentate, a whole broth fermentate extract obtained from a Bacillus velezensis fermentate, and any one combination thereof.

[0094] In one embodiment, the skin care composition is a composition for providing at least one skin care benefit described herein, wherein the Bacillus velezensis fermentate extract, or fraction thereof, is obtained from a fermentate of B. velezensis selected from the group consisting of a B. velezensis 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 of B. velezensis E04 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, a B. velezensis 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 of B. velezensis E04_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099, and any one combination thereof.

[0095] 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 velezensis 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, promoting skin repair, and any one combination thereof, wherein said skin barrier strengthening and / or said improving skin barrier function and / or said skin moisturization, occurs through increased tight junctions, increased desmosomes, increased cell-cell adhesion, increased cornification, and any one combination thereof.

[0096] In one aspect, the increased tight junctions occurs through an increased expression of a gene selected from the group consisting of CLDN5 (Gene ID 7122), JAML (Gene ID 120425), TJP3 (Gene ID 27134), CLDN9 (Gene ID 9080), CLDN4 (Gene ID 1364), CLDN16 (Gene ID 10686), CLDN7 (Gene ID 1366), OCLN (Gene ID 100506658), CLDN1 (Gene ID 9076), TJP2 (Gene ID 9414), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is identical to said skin care composition except for the absence of said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof.

[0097] In one aspect, the increased desmosomes occurs through an increased expression of a gene selected from the group consisting of DSG4 (Gene ID 147409), PPL (Gene ID 5493), EVPL (Gene ID 2125), DSC2 (Gene ID 1824), DSG3 (Gene ID 1830), DSP (Gene ID 1832), DSC3 (Gene ID 1825), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is identical to said skin care composition except for the absence of said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof.

[0098] In one aspect, the increased cell-cell adhesion occurs through an increased expression of a gene selected from the group consisting of PCDH1 (Gene ID 5097), CDH16 (Gene ID 1014), CNFN (Gene ID 84518), PCDHGB7 (Gene ID 56099), CDH26 (Gene ID 60437), CDHR1 (Gene ID 92211 ), PCDHB15 (Gene ID 56121 ), PCDHGB6 (Gene ID 56100), CADM4 (Gene ID 199731 ), NECTIN1 (Gene ID 5818), PCDHGA9 (Gene ID 56107), PCDHB14 (Gene ID 56122), PCDHGA11 (Gene ID 56105) and PCDHGA12 (Gene ID 26025), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is identical to said skin care composition except for the absence of said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof. In one aspect, the increased cornification occurs through an increased expression of a gene selected from the group consisting of FA2H (Gene ID 79152), TNFAIP6 (Gene ID 7130), TGM1 (Gene ID 7051 ), CERS3 (Gene ID 204219), LCE3D (Gene ID 84648), SCEL (Gene ID 8796), DMKN (Gene ID 93099), ELOVL4 (Gene ID 6785), CERS4 (Gene ID 79603), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is identical to said skin care composition except for the absence of said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof.

[0099] In one aspect the skin care composition described herein 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.

[0100] 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.

[0101] In one embodiment, the composition is a Bacillus velezensis strain variant fermentate, Bacillus velezensis strain variant fermentate extract, or fraction of said variant fermentate, of a B. velezensis strain variant, wherein said Bacillus velezensis strain variant is derived from a parental B. velezensis by knocking out a sporulation gene of said parental B. velezensis 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. velezensis strain, wherein said activity is selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof. In one embodiment, the composition is a Bacillus velezensis strain variant fermentate, Bacillus velezensis strain variant fermentate extract, or fraction of said variant fermentate, of a B. velezensis strain variant, wherein said Bacillus velezensis strain variant is derived from a parental B. velezensis by knocking out a sporulation gene of said parental B. velezensis 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. velezensis strain, wherein said activity is selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof, wherein the sporulation gene is spot IE.

[0102] In one embodiment, the composition is a Bacillus velezensis strain variant fermentate, Bacillus velezensis strain variant fermentate extract, or fraction of said variant fermentate, of a B. velezensis strain variant, wherein said Bacillus velezensis strain variant is derived from a parental B. velezensis by knocking out a sporulation gene of said parental B. velezensis 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. velezensis strain, wherein said activity is selected from the group consisting of 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 increased tight junctions, increased desmosomes, increased cell-cell adhesion, increased cornification, and any one combination thereof.

[0103] The skin care compositions, or any effective amount of said skin care composition, described herein can be formulated in a skin care product.

[0104] As used herein “skin care products” refer to products or formulations 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, foam, kits, or any one combinations thereof.

[0105] In one embodiment, the skin care product is a skin care product comprising the skin care composition described herein.

[0106] 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.

[0107] 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 velezensis fermentate extract, or fraction thereof.

[0108] In one embodiment, the skin care product is a skin care product comprising one or more dermatologically or skin care acceptable component and 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 B. velezensis 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, promoting skin repair, and any one combination thereof.

[0109] 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 a Bacillus velezensis fermentate extract, or fraction thereof, obtained from a fermentate of a B. velezensis selected from the group consisting of a B. velezensis 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 of B. velezensis E04 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, a B. velezensis 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 of 8. velezensis E04_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099, and any one combination thereof.

[0110] In one embodiment, the skin care product is a skin care product, wherein said skin care product comprises 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 a skin care composition described herein on a weight basis relative to a total weight of said skin care product.

[0111] 11 b. In one embodiment, the skin care product is a skin care product, wherein said skin care product comprises 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 a skin care composition described herein on a volume basis relative to a total volume of said skin care product.

[0112] In one aspect the skin care product is formulated for topical administration. In one aspect the skin care product is formulated for topical administration to the skin or scalp.

[0113] In one embodiment, the skin care product is a skin care product described herein 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.

[0114] 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.

[0115] 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).

[0116] Constituents of such vehicles may comprise water, aqueous buffer solutions, non- aqueous 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).

[0117] 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.

[0118] 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 (paraffin), 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.

[0119] 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).

[0120] A skin care product includes a cleansing / 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).

[0121] 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.

[0122] 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. "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.

[0123] The term "aqueous" refers to a formulation that contains water or that becomes water-containing following application to the skin or mucosal tissue.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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).

[0128] The dermatologically acceptable carrier may be a moisturizer formulation containing at least one emulsifiers, at least one surfactant, or any combination thereof.

[0129] 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.

[0130] 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 treatment 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. 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.

[0131] 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. 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.

[0132] 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 providing at least one skin care benefit as described herein.

[0133] In one aspect the kit is a kit comprising an effective amount of a Bacillus velezensis fermentate, a Bacillus velezensis fermentate extract, and / or fraction thereof for providing at least one skin care benefit selected from the group consisting of 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.

[0134] Methods for providing at least one skin care benefit

[0135] The skin care compositions and skin care products described herein can be used in methods for providing at least one skin care benefit. In one aspect the at least one skin care benefit is selected from the group consisting of 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), promoting skin repair, and any one combination thereof.

[0136] 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 described herein to the subject’s skin or scalp.

[0137] 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 of said subject, wherein said composition comprises an effective amount of a B. velezensis 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, promoting skin repair, and any one combination thereof.

[0138] 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 composition comprising an effective amount of a B. velezensis 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 skin barrier strengthening, improving skin barrier function, skin moisturization, promoting skin repair, and any one combination thereof.

[0139] In one aspect, 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 composition comprising an effective amount of a B. velezensis 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 skin barrier strengthening, improving skin barrier function, skin moisturization, promoting skin repair, and any one combination thereof, wherein the Bacillus velezensis fermentate extract, or fraction thereof, is obtained from a fermentate of B. velezensis selected from the group consisting of a B. velezensis 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 of B. velezensis E04 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, a B. velezensis 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 of B. velezensis E04_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099, and any one combination thereof. 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 of any preceding claim, to the skin or scalp of said subject.

[0140] 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 comprising an effective amount of a B. velezensis fermentate extract, or fraction thereof, to the skin or scalp of said subject.

[0141] In one embodiment, the method is 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 claim, to the skin or scalp of said subject.

[0142] In one embodiment, the method is 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 B. velezensis fermentate extract, or fraction thereof, to the skin or scalp of said subject.

[0143] In one embodiment, the method is 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.

[0144] In one embodiment, the method is a method for skin moisturization in a subject, the method comprising topically administering a skin care composition comprising an effective amount of a B. velezensis fermentate extract, or fraction thereof, to the skin or scalp of said subject.

[0145] 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%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% up to 50% of a skin care composition described herein on a weight basis relative to a total weight of said skin care product. 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%,

[0146] 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%,

[0147] 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% up to 50% of a skin care composition described herein on a volume basis relative to a total volume of said skin care product

[0148] Methods for increasing the activity of a Bacillus velezensis strain fermentate extract and compositions thereof

[0149] Methods for increasing the activity of a Bacillus velezensis strain fermentate extract for providing at least one skin care benefit are disclosed herein.

[0150] The inventors have unexpectedly observed that when a sporulation gene, such as but not limiting to SpollE, is knocked out in a parental B. velezensis strain (such as but not limiting to B. velezensis E04) to produce a B. velezensis variant strain (such as but not limiting to B. velezensis E04_spo), an increased activity of the fermentate, fermentate extract or fraction thereof derived from said B. velezensis variant strain was observed when compared to the activity of the fermentate, fermentate extract or fraction thereof from the parental B. velezensis strain, wherein said activity is selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

[0151] In one embodiment, the method is a method for increasing the activity of a Bacillus velezensis strain fermentate extract for providing at least one skin care benefit selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof, the method comprising: (a) providing a parent Bacillus velezensis 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 skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof; (b) modifying said parent Bacillus velezensis strain by knocking out at least one sporulation gene from the genome of said Bacillus velezensis strain, thereby producing a variant strain that is unable to form spores; and, (c) producing a fermentate extract of said Bacillus velezensis variant strain, wherein said fermentate extract of said Bacillus velezensis variant strain has an increased activity for providing at least one skin care benefit selected from the group consisting of 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 velezensis strain.

[0152] In one embodiment, the method is a method for increasing the activity of a Bacillus velezensis strain fermentate extract for providing at least one skin care benefit selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof, the method comprising: (a) providing a parent Bacillus velezensis 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 skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof; (b) modifying said parent Bacillus velezensis strain by knocking out at least one sporulation gene from the genome of said Bacillus velezensis strain, thereby producing a variant strain that is unable to form spores, wherein said at least one sporulation gene is SpollE and, (c) producing a fermentate extract of said Bacillus velezensis variant strain, wherein said fermentate extract of said Bacillus velezensis variant strain has an increased activity for providing at least one skin care benefit selected from the group consisting of 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 velezensis strain.

[0153] General definitions The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety.

[0154] In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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 to provide at least one skin care benefit.

[0159] Administering one or more microorganism (microbial strain), fermentate, fermentate extract or fraction 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), fermentate, fermentate extract or fraction 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.

[0160] 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.

[0161] 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”.

[0162] 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.

[0163] 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.

[0164] As used herein, the term “effective amount” refers to the amount sufficient to obtain the desired effect. A desired effect includes skin barrier strengthening, improving skin barrier function, skin moisturization, promoting skin repair, and any one combination thereof.

[0165] 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 condition or and / or one or more of its attendant symptoms or barring a subject from acquiring or reacquiring a condition or reducing a subject’s risk of acquiring or reacquiring a condition or one or more of its attendant symptoms.

[0166] 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%.

[0167] 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.

[0168] 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.

[0169] 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., 7th Edition, 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.

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] In one aspect the skin cells described herein are mammalian skin cells, such as human or animal skin cells.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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 velezensis 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 (protecting the skin against dehydration by maintaining, restoring and / or improving skin moisturization), promoting skin repair, and any one combination thereof. la. 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 velezensis 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 (protecting the skin against dehydration by maintaining, restoring and / or improving skin moisturization), promoting skin repair, and any one combination thereof. l b. The skin care composition of embodiment 1 , wherein said skin barrier strengthening comprises administering the composition to the subject’s skin or scalp to strengthen the skin barrier of said subject. lc. The skin care composition of embodiment 1 , 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. ld. The skin care composition of embodiment 1 , wherein said skin moisturization comprises administering the composition to the subject’s skin or scalp to moisturize the skin or scalp of said subject. le. The skin care composition of embodiment 1 , wherein said promoting skin repair comprises administering the composition to the subject’s skin or scalp to promote skin repair of said subject. lf. The skin care composition of embodiment 1 or 1 e, wherein the composition promotes skin repair through production of flagellin. le. The skin care composition of any preceding embodiments, wherein said composition comprises an effective amount of a Bacillus velezensis 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%, 35.0%, 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 100% by weight relative to a total weight of said composition. lf. The skin care composition of any preceding embodiments, wherein said composition comprises an effective amount of a Bacillus velezensis 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%, 35.0%, 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 volume relative to a total volume of said composition.

[0179] 2. The skin care composition of embodiment 1 , wherein the Bacillus velezensis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus velezensis fermentate, a whole broth fermentate extract obtained from a Bacillus velezensis fermentate, and any one combination thereof. 2b. The skin care composition of embodiment 1 , wherein the Bacillus velezensis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a Bacillus velezensis fermentate, a cell pellet extract obtained from a Bacillus velezensis fermentate, a whole broth fermentate extract obtained from a Bacillus velezensis fermentate, and any one combination thereof.

[0180] 2c. The skin care composition of embodiment 1 , wherein the Bacillus velezensis fermentate extract, or fraction thereof, is obtained from a fermentate of B. velezensis selected from the group consisting of a B. velezensis 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 of B. velezensis E04 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, a B. velezensis 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 of B. velezensis E04_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099, and any one combination thereof.

[0181] 3. 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 increased tight junctions, increased desmosomes, increased cell-cell adhesion, increased cornification, and any one combination thereof.

[0182] 4. The skin care composition of embodiment 3, wherein the increased tight junctions occurs through an increased expression of a gene selected from the group consisting of CLDN5 (Gene ID 7122), JAML (Gene ID 120425), TJP3 (Gene ID 27134), CLDN9 (Gene ID 9080), CLDN4 (Gene ID 1364), CLDN16 (Gene ID 10686), CLDN7 (Gene ID 1366), OCLN (Gene ID 100506658), CLDN1 (Gene ID 9076), TJP2 (Gene ID 9414), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is identical to said skin care composition except for the absence of said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof . 4b. The skin care composition of claim 3, wherein the increased tight junctions occurs through an increased expression of a gene selected from the group consisting of CLDN5 (Gene ID 7122), JAML (Gene ID 120425), TJP3 (Gene ID 27134), CLDN9 (Gene ID 9080), CLDN4 (Gene ID 1364), CLDN16 (Gene ID 10686), CLDN7 (Gene ID 1366), OCLN (Gene ID 100506658), CLDN1 (Gene ID 9076), TJP2 (Gene ID 9414), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is lacking said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof .

[0183] 5. The skin care composition of embodiment 3, wherein the increased desmosomes occurs through an increased expression of a gene selected from the group consisting of DSG4 (Gene ID 147409), PPL (Gene ID 5493), EVPL (Gene ID 2125), DSC2 (Gene ID 1824), DSG3 (Gene ID 1830), DSP (Gene ID 1832), DSC3 (Gene ID 1825), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is identical to said skin care composition except for the absence of said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof.

[0184] 5b. The skin care composition of embodiment 3, wherein the increased desmosomes occurs through an increased expression of a gene selected from the group consisting of DSG4 (Gene ID 147409), PPL (Gene ID 5493), EVPL (Gene ID 2125), DSC2 (Gene ID 1824), DSG3 (Gene ID 1830), DSP (Gene ID 1832), DSC3 (Gene ID 1825), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is lacking said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof.

[0185] 6. The skin care composition of embodiment 3, wherein the increased cell-cell adhesion occurs through an increased expression of a gene selected from the group consisting of PCDH1 (Gene ID 5097), CDH16 (Gene ID 1014), CNFN (Gene ID 84518), PCDHGB7 (Gene ID 56099), CDH26 (Gene ID 60437), CDHR1 (Gene ID 92211 ), PCDHB15 (Gene ID 56121 ), PCDHGB6 (Gene ID 56100), CADM4 (Gene ID 199731 ), NECTIN1 (Gene ID 5818), PCDHGA9 (Gene ID 56107), PCDHB14 (Gene ID 56122), PCDHGA11 (Gene ID 56105) and PCDHGA12 (Gene ID 26025), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is identical to said skin care composition except for the absence of said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof.

[0186] 6b. The skin care composition of embodiment 3, wherein the increased cell-cell adhesion occurs through an increased expression of a gene selected from the group consisting of PCDH1 (Gene ID 5097), CDH16 (Gene ID 1014), CNFN (Gene ID 84518), PCDHGB7 (Gene ID 56099), CDH26 (Gene ID 60437), CDHR1 (Gene ID 92211 ), PCDHB15 (Gene ID 56121 ), PCDHGB6 (Gene ID 56100), CADM4 (Gene ID 199731 ), NECTIN1 (Gene ID 5818), PCDHGA9 (Gene ID 56107), PCDHB14 (Gene ID 56122), PCDHGA11 (Gene ID 56105) and PCDHGA12 (Gene ID 26025), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is lacking said Bacillus velezensis fermentate extract, or fraction thereof.

[0187] 7. The skin care composition of embodiment 3, wherein the increased cornification occurs through an increased expression of a gene selected from the group consisting of FA2H (Gene ID 79152), TNFAIP6 (Gene ID 7130), TGM1 (Gene ID 7051 ), CERS3 (Gene ID 204219), LCE3D (Gene ID 84648), SCEL (Gene ID 8796), DMKN (Gene ID 93099), ELOVL4 (Gene ID 6785), CERS4 (Gene ID 79603), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is identical to said skin care composition except for the absence of said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof.

[0188] 7b. The skin care composition of embodiment 3, wherein the increased cornification occurs through an increased expression of a gene selected from the group consisting of FA2H (Gene ID 79152), TNFAIP6 (Gene ID 7130), TGM1 (Gene ID 7051 ), CERS3 (Gene ID 204219), LCE3D (Gene ID 84648), SCEL (Gene ID 8796), DMKN (Gene ID 93099), ELOVL4 (Gene ID 6785), CERS4 (Gene ID 79603), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is lacking said Bacillus velezensis fermentate extract, or fraction thereof. 8. The skin care composition of embodiment 1 , 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.

[0189] 9. Use of the skin care composition of any preceding embodiment in a cosmetic skin care product.

[0190] 9b. Use of an effective amount of the skin care composition of any preceding embodiment in a cosmetic skin care product.

[0191] 10. A skin care product comprising the skin care composition of any preceding embodiment and one or more dermatologically or skin care acceptable component. 10b. 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 velezensis fermentate extract, or fraction thereof.

[0192] 10c. A skin care product comprising one or more dermatologically or skin care acceptable component and 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 B. velezensis 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, promoting skin repair, and any one combination thereof.

[0193] 10b. 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 a Bacillus velezensis fermentate extract, or fraction thereof, obtained from a fermentate of a B. velezensis selected from the group consisting of a B. velezensis 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 of 8. velezensis E04 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, a B. velezensis 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 of B. velezensis E04_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099, and any one combination thereof.

[0194] 11 . The skin care product of embodiment 10, wherein said skin care product comprises 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.

[0195] 11 b. The skin care product of embodiment 10, wherein said skin care product comprises 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 volume basis relative to a total volume of said skin care product.

[0196] 12. The skin care product of embodiment 10 or embodiment 11 , wherein the product is formulated for topical administration.

[0197] 12b. The skin care product of embodiment 10 or 11 , wherein the product is formulated for topical administration to the skin or scalp.

[0198] 13. The skin care product of any one of embodiment 11 -12, 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.

[0199] 14. 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.

[0200] 14b 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 of said subject, wherein said composition comprises an effective amount of a B. velezensis 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, promoting skin repair, and any one combination thereof.

[0201] 14c. 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 B. velezensis 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 skin barrier strengthening, improving skin barrier function, skin moisturization, promoting skin repair, and any one combination thereof.

[0202] 15. The method of embodiment 14, wherein the skin care product is a 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.

[0203] 16. The method of any one of embodiment 14-embodiment 15, wherein the B. velezensis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a B. velezensis fermentate, a whole broth fermentate extract obtained from a B. velezensis fermentate, and any one combination thereof.

[0204] 16b. The method of any one of embodiment 14-embodiment 15, wherein the B. velezensis fermentate extract is selected from the group consisting of a cell free supernatant obtained from a B. velezensis fermentate, a cell pellet extract from a B. velezensis fermentate , a whole broth fermentate extract obtained from a B. velezensis fermentate, and any one combination thereof.

[0205] 16c. The method of any one of embodiment 14-embodiment 15, wherein the Bacillus velezensis fermentate extract, or fraction thereof, is obtained from a fermentate of B. velezensis selected from the group consisting of a B. velezensis 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 of 8. velezensis E04 deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, a B. velezensis 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 of B. velezensis E04_spo deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099, and any one combination thereof.

[0206] 16d. 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, 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.

[0207] 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.

[0208] 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 B. velezensis fermentate extract, or fraction thereof, to the skin or scalp of said subject.

[0209] 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.

[0210] 17b. 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 B. velezensis fermentate extract, or fraction thereof, to the skin or scalp of said subject.

[0211] 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. 18b. A method for skin moisturization in a subject, the method comprising topically administering a skin care composition comprising an effective amount of a B. velezensis fermentate extract, or fraction thereof, to the skin or scalp of said subject.

[0212] 19. A method for increasing the activity of a Bacillus velezensis strain fermentate extract for providing at least one skin care benefit selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof, the method comprising:

[0213] (a) providing a parent Bacillus velezensis 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 skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof;

[0214] (b) modifying said parent Bacillus velezensis strain by knocking out at least one sporulation gene from the genome of said Bacillus velezensis strain, thereby producing a variant strain that is unable to form spores; and,

[0215] (c) producing a fermentate extract of said Bacillus velezensis variant strain, wherein said fermentate extract of said Bacillus velezensis variant strain has an increased activity for providing at least one skin care benefit selected from the group consisting of 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 velezensis strain.

[0216] 19b. A method for increasing the activity of a Bacillus velezensis strain fermentate extract for providing at least one skin care benefit selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, promoting skin repair, and any one combination thereof in a subject in need thereof, the method comprising: (a) providing a parent Bacillus velezensis 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 skin barrier strengthening, improving skin barrier function, skin moisturization, promoting skin repair, and any one combination thereof in a subject in need thereof; (b) modifying said parent Bacillus velezensis strain by knocking out at least one sporulation gene from the genome of said Bacillus velezensis strain, thereby producing a variant strain that is unable to form spores; and, (c) producing a fermentate extract of said Bacillus velezensis variant strain, wherein said fermentate extract of said Bacillus velezensis variant strain has an increased activity for providing at least one skin care benefit selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, promoting skin repair, and any one combination thereof in a subject in need thereof, when compared to the activity of the fermentate extract of said parent Bacillus velezensis strain.

[0217] 19c. In one embodiment, the method is a method for increasing the activity of a Bacillus velezensis strain fermentate extract for providing at least one skin care benefit selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof, the method comprising: (a) providing a parent Bacillus velezensis 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 skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof in a subject in need thereof; (b) modifying said parent Bacillus velezensis strain by knocking out at least one sporulation gene from the genome of said Bacillus velezensis strain, thereby producing a variant strain that is unable to form spores, wherein said at least one sporulation gene is SpollE and, (c) producing a fermentate extract of said Bacillus velezensis variant strain, wherein said fermentate extract of said Bacillus velezensis variant strain has an increased activity for providing at least one skin care benefit selected from the group consisting of 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 velezensis strain. 20. A Bacillus velezensis strain variant fermentate, Bacillus velezensis strain variant fermentate extract, or fraction of said variant fermentate, of a B. velezensis strain variant, wherein said Bacillus velezensis strain variant is derived from a parental B. velezensis by knocking out a sporulation gene of said parental B. velezensis 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. velezensis strain, wherein said activity is selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, promoting skin repair, and any one combination thereof.

[0218] 20b. A Bacillus velezensis strain variant fermentate, Bacillus velezensis strain variant fermentate extract, or fraction of said variant fermentate, of a B. velezensis strain variant, wherein said Bacillus velezensis strain variant is derived from a parental B. velezensis by knocking out a sporulation gene of said parental B. velezensis 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. velezensis strain, wherein said activity is selected from the group consisting of 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 increased tight junctions, increased desmosomes, increased cell-cell adhesion, increased cornification, and any one combination thereof.

[0219] EXAMPLES

[0220] 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.

[0221] 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), “pL” 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.

[0222] EXAMPLE 1

[0223] Bacillus velezensis strains

[0224] Bacillus velezensis (B. velezensis) suitable for use in the present invention includes Bacillus velezensis 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 of Bacillus velezensis E04 (SEQ ID NO: 1 ) deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469.

[0225] Bacillus velezensis suitable for use in the present invention also includes Bacillus velezensis 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 of B. velezensis E04_spo (SEQ ID NO: 1 ) deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS151099, wherein the sporulation gene spollE has been knocked out. EXAMPLE 2

[0226] Fermentates of Bacillus velezensis

[0227] Bacillus velezensis 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. velezensis E04 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 24 hours.

[0228] Bacillus velezensis strains can also be 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 contains 1 ,47g Sodium Citrate 2H2O, 1.47 CaCI22 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 C0CI2 6 H2O, and 0.1 g Na2MoO4-2 H2O. The SUM medium can be adjusted to pH 7.3. Even though SUM medium can be used for growth medium of B. velezensis as described herein, B. velezensis strains can be grown in any other suitable media that can support growth. In addition, the incubation time may vary depending on the growth medium or method of 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 Bacillus velezensis fermentate extract.

[0229] EXAMPLE 3

[0230] Bacillus velezensis fermentate extracts

[0231] Extracts of the Bacillus velezensis fermentates were obtained as described below. Cell free supernatants of Bacillus velezensis fermentate

[0232] Cells were removed from the Bacillus velezensis 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.

[0233] Cell pellet extracts of Bacillus velezensis fermentate

[0234] A cell pellet extract from the Bacillus velezensis 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.

[0235] 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.

[0236] Combination of Cell free supernatant and Cell pellet extracts of Bacillus velezensis fermentate

[0237] Bacillus velezensis fermentate extracts can also be produced by combining the cell free supernatant fraction described above with the cell pellet extract described above.

[0238] Whole broth Bacillus velezensis fermentate extracts

[0239] To obtain a whole broth fermentate extract, the Bacillus velezensis 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 velezensis 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”).

[0240] EXAMPLE 4 Improvement of skin barrier function using Bacillus velezensis fermentate extract The ability of Bacillus velezensis fermentate extract to improve the skin barrier function & strengthen the skin barrier was evaluated. B. velezensis E04 strain (deposited at Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469) 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. velezensis E04 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 fermentate extract (cell free supernatant) was obtained by centrifugation and filter-sterilization of the fermentate.

[0241] 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. velezensis E04 fermentate extract was added such that the final fermentate extract concentration was 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 1 .

[0242] Table 1 . B. velezensis E04 fermentate extract (cell free supernatant) increases Transepidermal Electrical Resistance (TEER).

[0243] The results in Table 1 indicate that compared to untreated control, addition of 1 % B. velezensis E04 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.

[0244] EXAMPLE 5

[0245] Improvement of skin barrier function using fractions of B. velezensis fermentate extract

[0246] The ability of specific molecular weight fractions of B. velezensis fermentate extract to improve the skin barrier function and strengthen the skin barrier was evaluated. Fermentate extract (cell free supernatant) from B. velezensis E04 was generated as described in Example 4. Following centrifugation and filter sterilization of the fermentate to obtain the fermentate extract, 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: 100 kilodalton (kDa), 50 kDa, 30 kDa and 10 kDa. Fermentate extract 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. velezensis E04 molecular weight retentate and flow through were tested for the ability to improve skin barrier function using NHEK as performed in Example 1 . Results are shown in Table 2.

[0247] Table 2. Various B. velezensis E04 molecular weight fermentate extract fractions increase Transepidermal Electrical Resistance (TEER).

[0248] The results in Table 2 indicate that compared to untreated control, addition of 1 % B. velezensis E04 fermentate extract passed through a 100 kDa, 50 kDa, and 30 kDa molecular weight cut-off filter had a higher percent increase in TEER values, which is an indication of improved skin barrier function and strengthening of the skin barrier. EXAMPLE 6

[0249] Strengthening of the skin barrier by B. ve / ezens / s fermentate extract through tight junctions

[0250] The mechanism by which B. velezensis fermentate extract affects the keratinocyte barrier was evaluated. 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. velezensis E04 was generated as described in Example 4. Cells were incubated overnight in a 37°C incubator supplied with 5% CO2 and 95% humidity and then stimulated with B. velezensis E04 fermentate extract for 18 hours. Following stimulation with B. velezensis E04 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 either the Illumina NextSeq 1000 or Illumina NextSeq 2000 instrument. Table 3A-3B shows the expression of tight junction genes CLDN5 (Gene ID 7122, NCBI), JAML (Gene ID 120425), TJP3 (Gene ID 27134), CLDN9 (Gene ID 9080), CLDN4 (Gene ID 1364), CLDN16 (Gene ID 10686), CLDN7 (Gene ID 1366), OCLN (Gene ID 100506658), CLDN1 (Gene ID 9076) and TJP2 (Gene ID 9414) genes in NHEK of control and B. velezensis fermentate extract treated samples. Table 3A. B. velezensis E04 fermentate extract (cell free supernatant) increases the expression of tight junction genes CLDN5, JAML, TJP3, CLDN9, CLDN4, NHEK. Values are reported as Iog2 fold change Table 3B. B. velezensis E04 fermentate extract (cell free supernatant) increases the expression of tight junction genes CLDN16, CLDN 7, OCLN, CLDN1 and TJP2 NHEK. Values are reported as Iog2 fold change

[0251] The results presented in Tables 3A-3B demonstrate that B. velezensis E04 fermentate extract increases the expression of the CLDN5 (Gene ID 7122), JAML (Gene ID 120425), TJP3 (Gene ID 27134), CLDN9 (Gene ID 9080), CLDN4 (Gene ID 1364), CLDN16 (Gene ID 10686), CLDN7 (Gene ID 1366), OCLN (Gene ID 100506658), CLDN1 (Gene ID 9076) and TJP2 (Gene ID 9414) genes in NHEK indicating that B. velezensis E04 fermentate extract strengthens the keratinocyte barrier through increased tight junctions, and is therefore suitable for use in strengthening the skin barrier, improving the skin barrier function and skin moisturization.

[0252] EXAMPLE 7

[0253] Strengthening of the skin barrier by B. velezensis fermentate extract through desmosomes

[0254] The mechanism by which B. velezensis 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. velezensis E04 was generated as described in Example 1. Cells were incubated overnight in a 37°C incubator supplied with 5% CO2 and 95% humidity and then stimulated with B. velezensis E04 fermentate extract for 18 hours. Following stimulation with B. velezensis E04, 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 either the Illumina NextSeq 1000 or Illumina NextSeq 2000 instrument. Table 4 shows the expression of desmosome genes DSG4 (Desmoglein 4, Gene ID 147409), PPL (periplakin, Gene ID 5493), DSC2 (Desmocollin 2, Gene ID 1824), EVPL (envoplakin, Gene ID 2125), DSG3 (Desmogelin 3, Gene ID 1830), DSP (Desmoplakin, Gene ID 1832) and DSC3 (Desmocollin 3, Gene ID 1825) in NHEK of control and B. velezensis fermentate extract treated samples.

[0255] Table 4. B. velezensis E04 fermentate extract (cell free supernatant) increases the expression of desmosome genes DSG4, PPL, DSC2, EVPL, DSG3, DSP and DSC3 in NHEK. Values are reported as Ioq2 fold change

[0256] The results presented in Table 4 demonstrate that B. velezensis E04 fermentate extract increases the expression of the DSG4 (Gene ID 147409), DSC2 (Gene ID 1824), PPL (Gene ID 5493), DSG3 (Gene ID 1830), EVPL (Gene ID 2125), DSP (Gene ID 1832) and DSC3 (Gene ID 1825) genes in NHEK indicating that B. velezensis E04 strengthens the keratinocyte barrier through increased desmosomes, and is therefore suitable for use in strengthening the skin barrier, improving the skin barrier function and skin moisturization.

[0257] EXAMPLE 8 Strengthening of the skin barrier by B. ve / ezens / s fermentate extract through cellcell adhesion

[0258] The mechanism by which B. velezensis 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. velezensis E04 was generated as described in Example 1 . Cells were incubated overnight in a 37°C incubator supplied with 5% CO2 and 95% humidity and then stimulated with B. velezensis E04 fermentate extract for 18 hours. Following stimulation with B. velezensis E04, 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 either the Illumina NextSeq 1000 or Illumina NextSeq 2000 instrument. Table 5A-5B shows the expression of expression of cell-cell adhesion genes PCDH1 (Gene ID 5097), CDH16 (Gene ID 1014), CNFN (Gene ID 84518), PCDHGB7 (Gene ID 56099), CDH26 (Gene ID 60437), CDHR1 (Gene ID 92211 ), PCDHB15 (Gene ID 56121 ), PCDHGB6 (Gene ID 56100), CADM4 (Gene ID 199731 ), NECTIN1 (Gene ID 5818), PCDHGA9 (Gene ID 56107), PCDHB14 (Gene ID 56122), PCDHGA11 (Gene ID 56105) and PCDHGA12 (Gene ID 26025) in NHEK of control and B. velezensis fermentate extract treated samples.

[0259] Table 5A. B. velezensis E04 fermentate extract (cell free supernatant) increases the expression of cell-cell adhesion genes PCDH1 , CDH16, CNFN, PCDHGB7, CDH26, CDHR1 and PCDHB15 in NHEK. Values are reported as Iog2 fold change

[0260] Table 5B. B. velezensis E04 fermentate extract (cell free supernatant) increases the expression of cell-cell adhesion genes PCDHGB6, CADM4, NECTIN1 , PCDHGA9, PCDHB14, PCDHGA11 and PCDHA12 in NHEK. Values are reported as Iog2 fold change

[0261] The results presented in Table 5A-5B demonstrate that B. velezensis E04 increases the expression of the genes PCDH1 (Gene ID 5097), CDH16 (Gene ID 1014), CNFN (Gene ID 84518), PCDHGB7 (Gene ID 56099), CDH26 (Gene ID 60437), CDHR1 (Gene ID 92211 ), PCDHB15 (Gene ID 56121 ), PCDHGB6 (Gene ID 56100), CADM4 (Gene ID 199731 ), NECTIN1 (Gene ID 5818), PCDHGA9 (Gene ID 56107), PCDHB14 (Gene ID 56122), PCDHGA11 (Gene ID 56105) and PCDHGA12 (Gene ID 26025) in NHEK indicating that B. velezensis E04 fermentate extract strengthens the keratinocyte barrier through increased cell-cell adhesion, and is therefore suitable for use in strengthening the skin barrier, improving the skin barrier function and skin moisturization. EXAMPLE 9

[0262] Improvement of skin barrier by B. velezensis fermentate extract through cornification (cornified envelope)

[0263] The mechanism by which B. velezensis 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. velezensis E04 was generated as described in Example 4. Cells were incubated overnight in a 37°C incubator supplied with 5% CO2 and 95% humidity and then stimulated with B. velezensis E04 fermentate extract for 18 hours. Following stimulation with B. velezensis E04, 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 either the Illumina NextSeq 1000 or Illumina NextSeq 2000 instrument. Table 6A-6B shows the expression of genes FA2H (Gene ID 79152), TNFAIP6 (Gene ID 7130), TGM1 (Gene ID 7051 ), CERS3 (Gene ID 204219), LCE3D (Gene ID 84648), SCEL (Gene ID 8796), DMKN (Gene ID 93099), ELOVL4 (Gene ID 6785) and CERS4 (Gene ID 79603) in NHEK of control and B. velezensis fermentate extract treated samples. Table 6A. B. velezensis E04 fermentate extract (cell free supernatant) increases the expression of cornified envelope genes FA2H, TNFAIP6, TGM1 , CERS3, in NHEK. Values are reported as Iog2 fold change

[0264] Table 6B. B. velezensis E04 fermentate extract (cell free supernatant) increases the expression of cornified envelope genes LCE3D, DMKN, ELQVL4, CERS4 and SCEL in NHEK. Values are reported as Iog2 fold change

[0265] The results presented in Table 6A-6B demonstrate that B. velezensis E04 increases the expression of the genes FA2H (Gene ID 79152), TNFAIP6 (Gene ID 7130), TGM1 (Gene ID 7051 ), CERS3 (Gene ID 204219), LCE3D (Gene ID 84648), SCEL (Gene ID 8796), DMKN (Gene ID 93099), ELOVL4 (Gene ID 6785) and CERS4 (Gene ID 79603) in NHEK indicating that B. velezensis E04 strengthens the keratinocyte barrier through increased cornification, and is therefore suitable for use in strengthening the skin barrier, improving the skin barrier function and skin moisturization.

[0266] EXAMPLE 10

[0267] Knocking out B. velezensis sporulation gene spo / / E further enhances the ability of B. velezensis fermentate extracts to improve the skin barrier function.

[0268] B. velezensis strains contain genes associated with sporulation such as SpollE (SEQ ID NO: 2; Barak et al. 1996, Molecular Microbiology 19(5), 1047- 1060). In one aspect, elimination of spollE gene is desired, especially for B. velezensis production strains.

[0269] The ability of B. velezensis E04 sporulation spollE knockout (referred to as B. velezensis E04_spo, CBS 151099) fermentate extract to improve the skin barrier function was evaluated.

[0270] A CRISPR-Cas9 plasmid (referred to as pMY2-3) was constructed to enable deletion of the spollE locus from B. velezensis E04 (CBS147469) and, thereby, inhibits its ability to produce spores. The pMY2-3 plasmid comprised a Streptococcus pyogenes Cas9 endonuclease expression cassette as well as an editing template insert (SEQ ID NO: 3), containing the B. velezensis E04 homologous regions, the sgRNA (SEQ ID NO: 4), and a kanamycin resistance cassette. The pMY2-3 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 pMY2-3 using the GenomiPhi HY Ready-To-Go DNA Amplification Kit (Cytiva).

[0271] The B. velezensis E04 (CBS 147469) 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. velezensis E04 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 pMY2-3. A volume of 200 pL competent cells was mixed with 50 pL pMY2-3 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 pMY2-3. The isolates were screened for the deletion of spollE using Q5® PCR with primer BVspo-checkdel-F (SEQ ID NO: 5) and primer BVspo-checkdel-R (SEQ ID NO: 6). Colonies with the spollE deletion ( spollE) produced a PCR product of 2253 bp, whereas the wild-type strain containing the intact spollE gene produced a PCR product of 4743 bp. Deletions were confirmed via Sanger sequencing, and confirmed mutant isolates were stored as glycerol stocks at -80°C. The isolates were cured of pBL.comK and pMY2-3 by culturing them in tryptic soy broth (TSB) containing no antibiotics for three days, passaging into fresh TSB once every 24 hours, and then plating onto tryptic soy agar (TSA) containing no antibiotics. Colonies were selected from the TSA and patched onto plain LB agar plates containing no antibiotics, LB agar plates containing 20 ppm kanamycin, and LB agar plates containing 100 ppm spectinomycin. Cured isolates grew on plain LB agar and not LB containing kanamycin or spectinomycin.

[0272] Following genotypic confirmation of the spollE deletion, phenotypic analysis using heat treatment was conducted to confirm the loss of sporulation ability. B. velezensis E04 was grown in Difco sporulation medium (DSM) to promote the formation of spores (Nicholson, W.L. and Setlow, P. , 1990, Sporulation, germination, and outgrowth. In Molecular Biological Methods for Bacillus, edited by C.R. Harwood and S.M. Cutting, John Wiley & Sons, Chichester, UK, pp. 391- 450.). 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. velezensis E04, displayed growth at both room temperature (RT) and the 90°C treatment. Sporulationnegative isolates displayed growth at RT but not at 90°C. Sporulation-negative clones (also referred to as B. velezensis E04_spo strain) were stored as glycerol stocks at -80°C.

[0273] B. velezensis E04 (CBS147469, also referred to as B. velezensis E04 wild type (WT), and B. velezensis E04_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. velezensis E04 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.

[0274] 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 at 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. velezensis fermentate extract was added such that the final fermentate extract concentration was 0.5%. Following addition of fermentate extract, the transepidermal electrical resistance (TEER) was measured every day using an epithelial volt / ohm meter (EVOM). Media and ferment were refreshed every 2 days. Results are shown in Table 7.

[0275] Table 7. B. velezensis fermentate extract (cell free supernatant) of sporulation spollE knockout further increases Transepidermal Electrical Resistance TEER

[0276] The results in Table 7 indicate that addition of 0.5% of B. velezensis E04_spo fermentate extract (cell free supernatant) has a higher percent increase in TEER value when compared to the addition of 0.5% of B. velezensis E04, which is an indication of further improved barrier function, compared to an untreated control sample.

[0277] EXAMPLE 11

[0278] B. ve / ezens / s fermentate extracts for promoting skin repair.

[0279] The ability of B. velezensis fermentate extract to promote skin repair was evaluated. B. velezensis E04 strain 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. velezensis E04 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 fermentate extract (cell free supernatant) was obtained by centrifugation and filter-sterilization of the fermentate. HaCaT cells were thawed in DMEM supplemented with 10% FBS (complete media) and cultured in a T75 flask in a 37°C incubator supplied with 5% CO2 until reaching 90% confluency. After reaching confluency, HaCaT cells were removed from the flask using trypsin, centrifuged for 5 minutes at room temperature, resuspended in complete media and counted using a hemocytometer. HaCaT cells were subsequently seeded into 24 well plates and incubated in a 37°C incubator supplied with 5% CChfor 48hrs such the cells could reach 100% confluency. A scratch was then made in the confluent HaCaT cell layer, cells were washed with 1X PBS and then complete media was added back to the wells supplemented with or without 1 % B. velezensis E04 fermentate extract. The cells were incubated overnight in a 37°C incubator supplied with 5% CO2 and then imaged to assess repair / closure of the scratch. Results are shown in Table 8.

[0280] Table 8. B. velezensis E04 fermentate extract promotes skin repair.

[0281] The results in Table 8 demonstrate that treating keratinocytes with B. velezensis E04 fermentate extract increased closure (100% closure observed in Table 8) in the scratch test assay (smaller average scratch) indicating that B. velezensis E04 fermentate extract promotes skin repair.

[0282] EXAMPLE 12

[0283] B. velezensis fermentate extract fractions for promoting skin repair.

[0284] The ability of specific molecular weight fractions of B. velezensis fermentate extract (cell free supernatant) to promote skin repair was evaluated. A fermentate from B. velezensis E04 was generated using the method described in Example 11. A cell free supernatant fermentate extract was generated following centrifugation and filter sterilization of the fermentate. 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: 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 extracts that passed through the filters were saved and referred to as flow throughs (FT). Each B. velezensis E04 molecular weight retentate and flow through were tested for the ability to improve skin repair using HaCaT cells as performed in Example 11 . Results are sown in Table 9.

[0285] Table 9: Average scratch measurements of Bacillus velezensis E04 fermentate extracts and fractionations thereof.

[0286] The results in Table 9 demonstrate that treating keratinocytes with the B. velezensis E04 fermentate extract fraction containing components larger than 100 kDa increased closure in the scratch test assay (smaller average scratch measured) indicating that a component larger than 100 kDa produced by B. velezensis E04 promotes skin repair.

[0287] EXAMPLE 13

[0288] The B. velezensis fermentate extract bioactive flagellin mediates skin repair.

[0289] To identify the active component of B. velezensis that improves skin repair, the active molecular weight fraction (100 kDa retentate) was separated on a 4-12% polyacrylamide gel and the protein bands were visualized by Coomassie stain. Each separate band was excised from the gel and heated in 1X TBS overnight in order to extract the protein(s). The supernatant was collected and then processed for mass spectrometry analysis. Peptide fragments were assembled and BLASTx was used to identify all proteins in each corresponding band. Flagellin was identified as the most abundant protein in the skin repair active molecular weight fraction (100 kDa rententate) and therefore tested in the scratch assay.

[0290] B. velezensis flagellin was purified by collecting 1 .5 ml of overnight culture by centrifuging at 12K rpm for 2 minutes. The cells were washed 3 times with 1 ml of PBS and then suspended in 1 ml of PBS. The cell suspension was transferred to a microcentrifuge tube without beads. The tube was placed in a bead beater and agitated vigorously for 2 minutes. The supernatant containing the flagellin was collected after centrifugation at 12 K rpm for two minutes in a microfuge. To confirm flagellin was isolated, the supernatant was separated on a 4-12% polyacrylamide gel and protein bands identified using Coomassie. Only one band was visualized corresponding to 37 kDa, which is the size of a flagellin monomer. In native form, flagellin is assembled as an oligomer with molecular weights greater than 100 kDa.

[0291] Purified B. velezensis E04 flagellin was tested for the ability to improve skin repair using HaCaT cells as performed in Example 11 . Results are shown in Table 10.

[0292] Table 10: Average scratch measurements of Bacillus velezensis E04 flagellin

[0293] The results in Table 10 demonstrate that treating keratinocytes with the B. velezensis E04 purified flagellin increased closure in the scratch test assay (smaller average scratch measured) indicating that flagellin produced by B. velezensis E04 and present in the B. velezensis E04 fermentate extract promotes skin repair.

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 velezensis 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, promoting skin repair, and any one combination thereof.

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

3. The skin care composition of claim 1 , wherein said skin barrier strengthening and / or said improving skin barrier function and / or said skin moisturization occurs through increased tight junctions, increased desmosomes, increased cell-cell adhesion, increased cornification, and any one combination thereof.

4. The skin care composition of claim 3, wherein the increased tight junctions occurs through an increased expression of a gene selected from the group consisting of CLDN5 (Gene ID 7122), JAML (Gene ID 120425), TJP3 (Gene ID 27134), CLDN9 (Gene ID 9080), CLDN4 (Gene ID 1364), CLDN16 (Gene ID 10686), CLDN7 (Gene ID 1366), OCLN (Gene ID 100506658), CLDN1 (Gene ID 9076), TJP2 (Gene ID 9414), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is lacking said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof .

5. The skin care composition of claim 3, wherein the increased desmosomes occurs through an increased expression of a gene selected from the group consisting of DSG4 (Gene ID 147409), PPL (Gene ID 5493), EVPL (Gene ID 2125), DSC2 (Gene ID 1824), DSG3 (Gene ID 1830), DSP (Gene ID 1832), DSC3(Gene ID 1825), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is lacking said Bacillus velezensis fermentate extract, or fraction thereof.

6. The skin care composition of claim 3, wherein the increased cell-cell adhesion occurs through an increased expression of a gene selected from the group consisting of PCDH1 (Gene ID 5097), CDH16 (Gene ID 1014), CNFN (Gene ID 84518), PCDHGB7 (Gene ID 56099), CDH26 (Gene ID 60437), CDHR1 (Gene ID 92211 ), PCDHB15 (Gene ID 56121 ), PCDHGB6 (Gene ID 56100), CADM4 (Gene ID 199731 ), NECTIN1 (Gene ID 5818), PCDHGA9 (Gene ID 56107), PCDHB14 (Gene ID 56122), PCDHGA11 (Gene ID 56105) and PCDHGA12 (Gene ID 26025), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is lacking said Bacillus velezensis fermentate extract, or fraction thereof.

7. The skin care composition of claim 3, wherein the increased cornification occurs through an increased expression of a gene selected from the group consisting of FA2H (Gene ID 79152), TNFAIP6 (Gene ID 7130), TGM1 (Gene ID 7051 ), CERS3 (Gene ID 204219), LCE3D (Gene ID 84648), SCEL (Gene ID 8796), DMKN (Gene ID 93099), ELOVL4 (Gene ID 6785), CERS4 (Gene ID 79603), and any one combination thereof, when compared to the expression of said gene caused by a control composition that is lacking said effective amount of said Bacillus velezensis fermentate extract, or fraction thereof.

8. The skin care composition of claim 1 , 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.

9. Use of the skin care composition of any preceding claim in a cosmetic skin care product.

10. A skin care product comprising the skin care composition of any preceding claim and one or more dermatologically or skin care acceptable component.11 . The skin care product of claim 10, wherein said skin care product comprises 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.

12. The skin care product of claim 10 or claim 11 , wherein the product is formulated for topical administration.

13. The skin care product of any one of claim 11 -12, 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.

14. 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.

15. The method of claim 14, wherein the skin care product is a 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.

16. The method of any one of claim 14-claim 15, wherein the B. velezensis fermentate extract is selected from the group consisting of a cell free supernatantobtained from a B. velezensis fermentate, a whole broth fermentate extract obtained from a B. velezensis fermentate, and any one combination thereof.

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

20. A Bacillus velezensis strain variant fermentate, Bacillus velezensis strain variant fermentate extract, or fraction of said variant fermentate, of a B. velezensis strain variant, wherein said Bacillus velezensis strain variant is derived from a parental B. velezensis by knocking out a sporulation gene of said parental B. velezensis 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. velezensis strain, wherein said activity is selected from the group consisting of skin barrier strengthening, improving skin barrier function, skin moisturization, and any one combination thereof.

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