Fermented extracts and their use to provide skincare benefits

Abstract

This disclosure relates to skincare compositions, skincare products, and methods for providing at least one skincare effect in a subject that requires it. More specifically, this disclosure relates to methods for providing at least one skincare effect in a subject that requires it, including strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and promoting skin repair, and to compositions comprising Bacillus velezensis ferment, Bacillus velezensis ferment extract, or fraction thereof.

Description

[Technical Field]

[0001] This application claims the benefit of the specification of U.S. Provisional Patent Application No. 63 / 567133, filed March 19, 2024, which is incorporated herein by reference in its entirety.

[0002] This disclosure relates to skincare compositions, skincare products, and methods for providing at least one skincare effect in a subject that requires it. More specifically, this disclosure relates to methods for providing at least one skincare effect in a subject that requires it, including strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and promoting skin repair, and to compositions comprising Bacillus velezensis ferment, Bacillus velezensis ferment extract, or fraction thereof.

[0003] Reference to electronically submitted sequence listings The sequence listing, submitted electronically with this application as an XML file (name: 20240617_NB42137PCT_SequenceListing.xml, size: 12,460 bytes, creation date: June 17, 2024), constitutes a part of this application and is incorporated herein by reference in its entirety. [Background technology]

[0004] The skin not only protects organisms from dehydration but also acts as a barrier, shielding them from the intrusion of external substances, which are often harmful. The skin barrier is important for human life because it physically protects against external threats such as infectious pathogens, chemicals, systemic toxins, and allergens, internally promotes the maintenance of skin homeostasis, and protects against increased water loss from the body (Skin barrier function, 2016, T. Agner, editor, Karger, Basel, Switzerland, 164 pages, ISBN 978-3-318-05585-6).

[0005] Human skin consists of two main cell layers: the epidermis and the dermis. The epidermis is the outermost layer of skin and is mainly composed of terminally differentiated keratinocytes and lipids. Beneath the terminally differentiated keratinocytes are dividing, living keratinocytes. The outer layer of the epidermis (stratum corneum or cornea) is the part that comes into contact with the environment, and the specific structure of the stratum corneum protects the skin and stabilizes its own flexibility by binding with a certain amount of water (PMElias, Structure and Function of the Stratum Corneum Permeability Barrier, Drug Dev. Res. 13, 1988, 97-105). The skin barrier function largely depends on the structure and composition of the stratum corneum, which consists of flat, anucleated cells surrounded by a highly organized, 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 a permeable barrier against environmental challenges such as UV radiation, heat, chemicals, pollution, and pathogens, including bacteria, fungi, parasites, and viruses. It also protects the body from uncontrolled internal water evaporation and maintains hydration balance and skin metabolism.

[0006] The stratified epithelium of the skin is composed of multiple layers of keratinocytes in various differentiated states. These layers, from the topmost to the bottommost, are generally called the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale. In summary, these layers provide multiple functionalities to the skin, and their primary function is to provide a physical barrier against the external environment and microorganisms (Hogan et al., 2012, Journal of Allergy 2012:901940:1-7).

[0007] As keratinocytes differentiate, the cells express numerous proteins, enzymes, and lipids involved in the formation of the stratum corneum, the skin's frontline barrier. Essential features of the stratum corneum that contribute to the integrity of the skin barrier are cross-linked structural proteins, lipids, and tightly linked cell-cell connections. Genetic mutations that impair the production of these components are involved in many skin disorders, demonstrating that all barrier components are important for maintaining skin health (Eckert, R.L. and EARorke, 1989, Environmental Health Perspectives 80:109-116; O'Regan GM, 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, FrontCell.Neurosci.2019:13; Lee J.Y.Wand J.A.C.Grath, 2021, British Journal of Dermatology 184(4):596-605).

[0008] Adjacent, tightly linked keratinocytes in the stratum corneum are a key feature that maintains the integrity of the skin barrier, thereby limiting water loss and preventing the transfer 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)). The expression of a series of transmembrane proteins is one strategy that keratinocytes use to link together. Three important transmembrane systems that ensure adjacent keratinocytes are tightly linked together are desmosomes, cadherins (Cdh), and protocadherins (Pcdh). Desmosomes, which are intracellular adhesion junctions that link the intermediate filaments of adjacent cells, are composed of key proteins such as desmoglein (Dsg), desmocolin (Dsc), and desmoplankin (DP). Disruption of desmosome formation can lead to loss of skin barrier integrity (Lee JYWand JAMcGrath, 2021, British Journal of Dermatology 184(4):596-605, Johnson J. et al., 2014, Cold Spring Harbor Perspectives in Medicine 4:(11)). Cadherins and their subfamily protocadherins, like desmosomes, are important intracellular adhesion junctions that link actin filaments of adjacent cells. Both belong to a large class of proteins, and protocadherins are further distinguished by three subclasses: alpha, beta, and gamma. Disruption of cadherin and protocadherin function can result in loss of epithelial barrier integrity and the 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 CL, et al., 2008, PNAS 105(40):15405-15410).

[0009] Keratinocytes in the stratum corneum form a special membrane called the cornified envelope through the process of differentiation. The cornified envelope is mainly composed of cross-linked structural proteins and lipids and contributes to maintaining the integrity of the skin barrier. Important proteins that confer functionality to the cornified envelope include filaggrin, involucrin, loricrin, late envelope protein (LCE), and sielin (SCEL). Enzymes are required to cross-link proteins such as involucrin and loricrin, with transglutimans (TGM) being the most common. Ceramides and long-chain fatty acids are among the most abundant lipids in the stratum corneum and help provide a hydrophobic barrier to maintain water and moisture homeostasis. The enzymes important for the synthesis of these lipids are a class called ceramide synthase (CERS) and elongase of very long-chain fatty acids (ELOVL). Since ceramide requires the addition of sphingolipids to fatty acids, enzymes such as FA2H and fatty acid 2-hydroxylase are necessary for sphingolipid production (Proksch E., et al., 2008, Experimental Dermatology 17:1063-1072, Uchida Y., et al., 2007, Journal of Biological Chemistry 282(18):13211-13219).

[0010] An important function of the skin barrier is to maintain moisture, which involves carefully balancing the moisture content (Proksch E., et al., 2008, Experimental Dermatology 17:1063-1072). In general, the skin barrier prevents excessive moisture loss and absorption, ensuring that the skin remains moist and does not dry out. One important component of the epidermal layer that maintains moisture is hyaluronic acid (HA) (Papakonstantinou E., et al., 2012, DermatoEndicrinolgy 4(3):253-258). To become fully effective, HA is cross-linked, a process mediated by enzymes such as TNFAIP6 (Evrard C., et al., 2021, JID Innovations 1:100054). Damage to the physical barrier caused by abrasive or inflammatory skin diseases, including but not limited to eczema, psoriasis, and atopic dermatitis, can disrupt the balance of moisture regulation. Therefore, when the physical skin barrier is damaged, the proper functioning of cellular processes necessary to restore the skin barrier to its intact form is essential. Dryness and chronic inflammation can occur as a result of chronic exposure to the layers beneath the epidermis and dermis, as well as from persistent exposure to microbial pathogens and environmental pollutants, as manifested in conditions such as eczema, psoriasis, atopic dermatitis, contact dermatitis, burns and complications from diabetes, vasculitis, and ulcers resulting from ischemic injury (Proksch E., et al., 2008, Experimental Dermatology 17:1063-1072). A common method for measuring skin barrier integrity and, in relation to skin moisturizing, is to measure transepidermal water loss (TEWL). TEWL measurements indicate the amount of water lost from the skin surface. A higher TEWL value indicates greater water loss and therefore more severe skin dryness (Proksch E., et al., 2008, Experimental Dermatology 17:1063-1072). Measuring the electrical resistance of the skin barrier is another way to assess the integrity of the skin barrier. Higher resistance values ​​correlate with better barrier integrity, while lower resistance values ​​correlate with lower barrier integrity.TEWL values ​​are known to correlate with electrical resistance measurements in laboratory environments (Guth K., et.al., 2015, Toxicology In Vitro 29:113-123). Therefore, lower TEWL values ​​may be reflected by higher electrical resistance values, thus indicating an intact barrier that leads to minimal moisture loss.

[0011] Current treatments for most skin disorders directly target either inflammation or associated microorganisms, and inhibiting either of these components allows the skin to recover and form an intact barrier. For example, cytokine inhibition using biologics such as monoclonal antibodies in skin disorders such as psoriasis and atopic dermatitis can normalize the expression of filaggrin and other barrier components (Jeon C., et.al., 2017, Hum.Vaccin.Immunother. 13(10):2247-2259, Bieber T., 2020, European Journal of Allergy and Clinical Immunology 75(1):54-62).

[0012] The skin is also a habitat for diverse microbial communities, the majority of which are symbiotic (non-pathogenic commensal) or transient (temporarily commensal) organisms. When interacting with pathogens, only the microorganisms benefit, while the host ultimately suffers harm. Many skin pathogens that inhabit the skin can typically be seen as symbiotic organisms, but disruptions in the microbiome (or microbial imbalances), host genetic mutations, and immune status can trigger a shift from symbiotic to pathogenic forms (Findley, K. and Grice, EA, The Skin Microbiome: A Focus on Pathogens and Their Association with Skin Disease. PLoS Pathog. 2014, 10).

[0013] The epidermis constitutes the outermost layer of skin tissue and forms the actual protective outer layer against the environment. The outer layer of the epidermis (stratum corneum or stratum corneum) is the part that comes into contact with the environment, and the specific structure of the stratum corneum protects the skin and stabilizes its own flexibility by holding a certain amount of water (PMElias, Drug Dev. Res. 13, 1988, 97-105). [Overview of the project] [Problems that the invention aims to solve]

[0014] There is still a need to find methods and skincare compositions for strengthening the skin barrier of the skin or scalp, improving skin barrier function, and moisturizing the skin. [Means for solving the problem]

[0015] This disclosure relates to skincare compositions, skincare products, and methods for providing at least one skincare effect in subjects requiring it. More specifically, this disclosure relates to methods for providing at least one skincare effect in subjects requiring it, including strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and promoting skin repair, and to compositions comprising Bacillus velezensis ferment, Bacillus velezensis ferment extract, or fraction thereof. Furthermore, methods for increasing the activity of Bacillus velezensis strain ferment extracts to provide at least one skincare effect are disclosed herein.

[0016] The inventors unexpectedly observed that a fermented extract (cell-free supernatant) of Bacillus velezensis (including, but not limited to, B. velezensis E04 and B. velezensis E04_spo) strengthens the skin barrier, improves skin barrier function, and promotes skin repair.

[0017] In one embodiment, the skincare composition is a skincare composition for providing at least one skincare effect to a subject in need thereof, the composition comprising an effective amount of Bacillus velezensis ferment extract or fraction thereof, the at least one skincare effect being selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof.

[0018] In one embodiment, the skincare composition is a composition for providing at least one skincare effect to a subject in need thereof, the composition comprising an effective amount of Bacillus velezensis ferment extract or a fraction thereof, the at least one skincare effect being selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof, the Bacillus velezensis ferment extract being selected from the group consisting of cell-free supernatant obtained from Bacillus velezensis ferment, whole broth ferment extract obtained from Bacillus velezensis ferment, and any one combination thereof.

[0019] In one embodiment, the skincare composition is a skincare composition for providing at least one skincare effect in a subject requiring it, the composition comprising an effective amount of Bacillus velezensis ferment extract or fraction thereof, the at least one skincare effect being selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any combination thereof, wherein the strengthening of the skin barrier, and / or the improvement of skin barrier function, and / or the moisturizing of the skin occurs through an increase in tight junctions, an increase in desmosomes, an increase in cell-cell adhesion, an increase in keratinization, and any combination thereof.

[0020] In one aspect, the promotion of skin repair occurs through the proliferation and migration of keratinocytes.

[0021] In one aspect, the skin care composition described herein further comprises an additional compound selected from the group consisting of a fragrance, an excipient, a preservative, a pH adjuster, an aqueous carrier, an alcohol carrier, a skin softener, a coagulant, a humectant, an emulsifier, a solubilizer, a surfactant, a thickener, a salt, and any combination of any one of them.

[0022] In one embodiment, the composition is a fermentation product of a Bacillus velezensis strain variant, a fermentation extract of a Bacillus velezensis strain variant or a fraction of the fermentation product of a B. velezensis strain variant, wherein the Bacillus velezensis strain variant is obtained from the parental B. velezensis by knocking out the sporulation gene of the parental B. velezensis, and the fermentation product, the fermentation extract or the fraction of the fermentation product has increased activity compared to the activity of the fermentation product, fermentation extract or fraction thereof derived from the parental B. velezensis strain, and the activity is selected from the group consisting of enhancing the skin barrier, improving the skin barrier function, skin moisturization, and any combination of any one of them.

[0023] In one embodiment, the skin care product is a skin care product comprising a skin care composition for providing at least one skin care effect in a subject in need thereof, the composition comprising an effective amount of a Bacillus velezensis fermentation extract or a fraction thereof, and the at least one skin care effect is selected from the group consisting of enhancing the skin barrier, improving the skin barrier function, skin moisturization, promoting skin repair, and any combination of any one of them.

[0024] In one embodiment, the method is a method for providing at least one skincare effect to a subject in need thereof, the method comprising topically administering a skincare composition or a skincare product containing the composition to the skin of the subject, the composition comprising an effective amount of B. velezensis ferment extract or a fraction thereof, and the at least one skincare effect is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof.

[0025] In one embodiment, the method is a method for increasing the activity of a fermented extract of the Bacillus velezensis strain to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject in need. [Modes for carrying out the invention]

[0026] The features and advantages of this disclosure will be more readily understood by those skilled in the art by reading the following detailed description. For clarity, it should be understood that certain features of this disclosure described above and below in relation to separate embodiments may be provided in a single element or in combination. Conversely, for brevity, various features of this disclosure described in relation to a single embodiment may be provided separately or in any secondary combination. It will be understood below that embodiments mentioned in relation to one broad aspect of the invention are equally applicable to each of the other broad aspects of the invention described above. Unless otherwise indicated in the context, it will be further understood that the embodiments described below may be combined.

[0027] Microorganisms, fermented products, and fermented extracts As used herein, “microorganism” or “microbe” refers to bacteria, fungi, viruses, protozoa, archaea, and other microorganisms or microscopic organisms.

[0028] In some embodiments, Bacillus velezensis cells suitable for use in the present invention may be subjected to treatments to eliminate their replication ability, such as exposure to heat, drying, gamma irradiation, or ultraviolet irradiation. Non-replicating microorganisms suitable for use in the present invention may be dead cells or living cells that have lost the ability to divide. Non-replicating microorganisms suitable for use in the present invention may be intact cells or cells that have been partially or completely subjected to cytolysis. In some embodiments, non-replicating cells may comprise a mixture of intact cells and cytolysates.

[0029] Microorganisms suitable for use in the present invention may be included in the compositions according to the present invention in a living, semi-active or inactive form, or dead form. In the spirit of the present invention, "inactivated" or "dead" microorganisms are those that can no longer form colonies in a culture. Dead or inactivated microorganisms may be intact or have their cell membranes destroyed. Dead or inactivated microorganisms may be obtained by any method known to those skilled in the art.

[0030] In one embodiment, a strain of Bacillus velezensis is mentioned as a microorganism suitable for use in the present invention.

[0031] In some embodiments, suitable microorganisms for use in the present invention include bacterial strains having a 16S ribosomal RNA sequence that exhibits 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 the 16S ribosomal RNA sequence (SEQ ID NO: 1) of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under the number CBS147469.

[0032] The 16S ribosomal RNA sequence of Bacillus velezensis E04 is as follows: [ka]

[0033] In some embodiments, suitable microorganisms for use in the present invention include B. velezensis E04 with a 16S ribosomal RNA sequence (SEQ ID NO: 1) containing 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%, and 99.92%. Examples include bacterial strains with 16S ribosomal RNA sequences exhibiting sequence similarity of 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100%, in which the sporogenesis gene spoIIE is knocked out, which is called B. velezensis E04_spo and deposited with the Westerdijk Fungal Biodiversity Institute (WFDB) under the number CBS151099.

[0034] In one aspect of the present invention, a fermented product is provided.

[0035] As used herein, the term “fermentation product” should be understood as a composition (compound mixture) produced by growing live microorganisms (microbial strains) in a nutrient medium. Fermentation products may include cell population components derived from the microorganisms, unused medium components, and metabolites (i.e., unused substrates and / or fermentation end products). As used herein, “cell population components” refers to any mixture of proteins, lipids (i.e., membranes), carbohydrates, extracellular polysaccharides, metabolites, etc., from the grown microorganisms. For example, when microorganisms grow, new cells are produced that generally include additional cell populations such as, but are not limited to, cell membranes, nucleic acids (i.e., DNA and / or RNA), intracellular structures, polysaccharides, and proteins (i.e., membrane-bound, secretory, and / or intracellular).

[0036] Examples of fermented products used in the present invention include those derived from the microorganism Bacillus velezensis.

[0037] The growth medium used to prepare the fermented product is any medium containing the necessary nutrients suitable for the growth of microorganisms suitable for use in the present invention. Suitable nutrients include, but are not limited to, amino peptides, peptides, yeast extracts, salts, sugars, hydrocarbons, and / or vitamins. The medium can be based on dairy products such as milk, grains, fruits, and / or vegetables.

[0038] In one embodiment, the fermented product used in the present invention is a fermented product derived from the microorganism Bacillus velezensis, which is registered under the number CBS147469 at Westerdijk Fungal Biodiversity. The selection is made from Bacillus velezensis specimens having 16S ribosomal RNA sequences that exhibit sequence similarity of 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% to the 16S ribosomal RNA sequence (SEQ ID NO: 1) of B. velezensis E04 deposited at the World Food Database (WFDB).

[0039] In one embodiment, the fermented product used in the present invention is a fermented product derived from the microorganism Bacillus velezensis, which is registered under the number CBS151099X at Westerdijk Fungal Biodiversity. The selection is made from Bacillus velezensis species that have a 16S ribosomal RNA sequence showing 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 the 16S ribosomal RNA sequence (SEQ ID NO: 1) of B. velezensis E04_spo deposited at the World Food Database (WFDB).

[0040] To obtain an effective amount of active substance in the ferment, the ferment can be further concentrated before being included in the composition. The fermentation extract can be produced from the ferment described herein, and the ferment can be spray-dried or freeze-dried before being included in the composition.

[0041] It will be apparent that the fermented product can be used directly in the compositions and methods of the present invention, or that one or more fractions or extracts containing the active substance from the fermented product can be isolated from the fermented product by any suitable means prior to use.

[0042] In one aspect of the present invention, a fermented extract is provided. Examples of fermented extracts used in the present invention include those derived from the microorganism Bacillus velezensis.

[0043] As used herein, the term “fermentation extract” refers to an extract or fraction from a fermentation product produced by growing a living microorganism (microbial strain) in a nutrient medium as described above. In one embodiment, the fermentation extract is an extract of a fermentation product produced by growing the species Bacillus velezensis.

[0044] In one embodiment, the fermentation extract is the cell-free supernatant of the ferment. As used herein, “cell-free supernatant,” “fermentation supernatant,” “cell-free ferment,” or “fermentation filtrate” are used interchangeably and refer to a fermentation extract substantially free of viable cells, e.g., the supernatant of a cell culture of at least one microorganism from which cells have been removed. Cells may be removed from the cell culture by any method known in the art, and it is understood that such cell removal (e.g., by centrifugation, filtration) may still result in a cell-free supernatant that may contain trace amounts of cells or cell debris. Methods for separating cells from growth media are well known in the art and can utilize physical methods, e.g., centrifugation, filtration, ultrafiltration, tangential flow filtration, volumetric filtration, or reverse osmosis to produce cell pellets and culture supernatants. Alternatively or in addition, the separation method may be ligand-based and include, for example, an antibody that specifically binds to Bacillus velezensis. The antibody may be conjugated to a solid support such as magnetic beads. In one embodiment, the cell-free supernatant is obtained by filtering or centrifugation of the culture medium in which Bacillus velezensis cells were cultured.

[0045] In one embodiment, the cell-free supernatant (also called the fermentation extract) is obtained by filtration or centrifugation of the Bacillus velezensis ferment.

[0046] In one embodiment, cells were pelletized (centrifuged at 4,000-8,000 × g), and the supernatant was passed through a 0.2 μM filter to remove cells from the Bacillus velezensis ferment to obtain an essentially cell-free supernatant.

[0047] In one embodiment, the fermentation extract for use in the present invention is a Bacillus velezensis fermentation extract consisting essentially of a cell-free ferment. The term “consistently of” in relation to the ferment includes that at least 90% of the ferment has a specified property (e.g., being a cell-free ferment). Preferably, at least 95% has the specified property. Preferably, at least 97% has the specified property. Preferably, at least 99% has the specified property. In some embodiments, at least 100% has the specified property.

[0048] The fermentation extracts for use in the compositions, methods, and / or uses of the present invention may not substantially contain living Bacillus velezensis cells, and typically the living cells are 0 (or substantially 0) cells / mL in the ferment.

[0049] In another embodiment, the fermentation extract is an extract of a cell pellet obtained from a fermented product by pelletizing cells. The cell pellet can be obtained by centrifugation of the fermented product and removal of the cell-free supernatant.

[0050] In one embodiment, the cell pellet extract is obtained from a Bacillus velezensis ferment by pelletizing the cells (centrifugation at 4,000-8,000 × g), draining the supernatant, and leaving a portion of the cell pellet. The pellet is resuspended in acidic water (1 / 10 v / v) at pH 2.0-4.0, vortexed, pelletized by centrifugation (4,000-8,000 × g), and the extract is passed through a 0.2 μM filter. Starting with a cell pellet has the advantage that the volume of the resuspension (extract volume) can be determined to produce an effective amount of active extract, and / or it can be further concentrated to produce an effective amount of active extract. Furthermore, by resuspending the pellet in water or a non-fermented broth liquid, non-effective components of the fermented broth that may interfere with the active substances in the cell pellet extract are reduced or removed.

[0051] In another embodiment, a Bacillus velezensis fermentation extract is produced by combining the cell-free supernatant fraction described above with the cell pellet extract described above.

[0052] In yet another embodiment, the Bacillus velezensis fermentation extract is a whole broth fermentation extract prepared by first adjusting the pH of the ferment (whole fermentation broth) to 2.0-4.0, then pelletizing the insoluble cellular material, and optionally filtering the supernatant through a 0.2 μM filter to obtain a cell-free supernatant (also called whole broth fermentation extract). The fermentation extract may be further concentrated or purified before being included in a composition to obtain an effective amount of fermentation extract. The fermentation extract may be spray-dried or freeze-dried before being included in a composition.

[0053] Instead, Bacillus velezensis fermentation extract is a whole broth fermentation extract prepared by first adjusting the pH of the ferment (whole fermentation broth) to an alkaline pH, then pelletizing the insoluble cellular material, and optionally filtering the supernatant through a 0.2 μM filter to obtain a cell-free supernatant (also called "whole broth fermentation extract").

[0054] In one embodiment, the fermentation extract is obtained from a fermented product produced using a nutrient medium having a pH of 2 to 12.

[0055] In one embodiment, the fermentation extract is obtained from a fermented product whose pH has been adjusted to 2-12 before obtaining the fermentation extract.

[0056] It is understood that the production of fermented products and fermentation extracts may vary from batch to batch (between fermentations), resulting in different effects of the fermentation extracts. Therefore, batch-to-batch variability in the production of effective amounts of Bacillus velezensis fermentation extract may be observed.

[0057] In one embodiment, a Bacillus velezensis ferment, a fermentation extract, or a composition containing a fermentation extract is formulated in a dry or liquid formulation.

[0058] In one embodiment, a Bacillus velezensis ferment, fermentation extract, or composition containing a fermentation extract is formulated in at least one form selected from the group consisting of loose powder or compact powder, granules, liquid suspension or solution, spray solution, or any combination thereof.

[0059] In one embodiment, the granules are present in amounts of approximately 0.1% by weight, 0.2% by weight, 0.3% by weight, 0.4% by weight, 0.5% by weight, 0.6% by weight, 0.7% by weight, 0.8% by weight, 0.9% by weight, 1% by weight, 2% by weight, 3% by weight, 4% by weight, 5% by weight, 6% by weight, 7% by weight, 8% by weight, 9% by weight, 10% by weight, 11% by weight, 12% by weight, 13% by weight, 14% by weight, 15% by weight, 16% by weight, 17% by weight, 18% by weight, 19% by weight, 20% by weight, 21% by weight, 22% by weight, 23% by weight, 24% by weight, 25.0% by weight, 26% by weight, and 2% by weight, relative to the total weight of the granules. Contains 7% by weight, 28% by weight, 29% by weight, 30% by weight, 31% by weight, 32% by weight, 33% by weight, 34% by weight, 35.0% by weight, 36% by weight, 37% by weight, 38% by weight, 39% by weight, 40% by weight, 41% by weight, 42% by weight, 43% by weight, 44% by weight, 45% by weight, 46% by weight, 47% by weight, 48% by weight, 49% by weight, 50% by weight, 55% by weight, 60% by weight, 65% by weight, 70% by weight, 75% by weight, 80% by weight, 85% by weight, 90% by weight, 95% by weight, or up to 100% by weight of the Bacillus velezensis fermentation extract described herein.

[0060] The granules can be produced by any means known in the art, such as spray-drying the fermentation extract on a core or spray-drying the ferment to form the granules themselves, although these are not excluded. In one embodiment, the granules are easily dispersible layered granules. The layered granules may comprise a core surrounded by a coating layer containing at least one effective amount of Bacillus velezensis fermentation extract dispersed within a protective matrix, the core being water-soluble and rapidly soluble. The protective matrix may comprise at least one polyhydroxy compound and at least one phosphate compound.

[0061] In one embodiment, a Bacillus velezensis ferment, a Bacillus velezensis ferment extract, or a fraction thereof is effective in providing at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any combination thereof.

[0062] Strengthening or improving the skin barrier means that the skin (including, but not limited to, its permeability barrier) becomes "tighter / stronger" in some way, limiting or preventing the entry of compounds (e.g., microorganisms, contaminants), as well as limiting or preventing the amount of water that escapes, i.e., improving the skin barrier function.

[0063] In one embodiment, the fermentation extract or fraction thereof for use in the present invention comprises a fermentation extract or fraction thereof derived from the microorganism Bacillus velezensis, which is classified under the number CBS147469 by Westerdijk Fungal Biodiversity. The selection is made from Bacillus velezensis specimens having 16S ribosomal RNA sequences that exhibit sequence similarity of 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% to the 16S ribosomal RNA sequence (SEQ ID NO: 1) of B. velezensis E04 deposited at the World Food Database (WFDB).

[0064] In one embodiment, the fermentation extract or fraction thereof for use in the present invention comprises a fermentation extract from the microorganism Bacillus velezensis, wherein the B. velezensis has the sporogenesis gene spoIIE knocked out, under the number CBS151099 by Westerdijk Fungal Biodiversity. The selection is made from B. velezensis strains that have a 16S ribosomal RNA sequence showing 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 the 16S ribosomal RNA sequence (SEQ ID NO: 1) of B. velezensis E04_spo deposited at the World Food Database (WFDB).

[0065] Skincare composition and skincare product for providing at least one skincare effect As used herein, the term “skincare composition” means a composition comprising at least one skincare-beneficial substance that can provide a skincare effect (skincare effect).

[0066] As used herein, the terms “skin care substance,” “activator,” or “bioactive substance” are used interchangeably and refer to Bacillus velezensis ferment, Bacillus velezensis ferment extract, or fraction thereof that may provide skin care (scalp care) effects.

[0067] In some embodiments provided herein, the skincare substance comprises an effective amount of Bacillus velezensis ferment, Bacillus velezensis ferment extract, or fraction thereof, wherein Bacillus velezensis is distributed under the number CBS147469 by Westerdijk Fungal Biodiversity. Westerdijk Fungal under the number CBS151099 has a 16S ribosomal RNA sequence that exhibits 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 the 16S ribosomal RNA sequence (SEQ ID NO: 1) of B. velezensis E04 deposited at the World Food Database (WFDB). The selection is made from B. velezensis strains having a 16S ribosomal RNA sequence that exhibits 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 the 16S ribosomal RNA sequence (SEQ ID NO: 1) of B. velezensis E04_spo deposited at the Biodiversity Institute (WFDB).

[0068] As used herein, the term “skin care effect” refers to the effect provided by a skin care effect substance (or a skin care composition and / or skin care product containing an effective amount of such skin care effect substance) when applied topically to the skin or scalp, such as a ferment, fermentation extract or fraction thereof. Unexpectedly, the inventors observed that a fermentation extract of Bacillus velezensis (cell-free fermentation supernatant) strengthens the skin barrier (improves the keratinocyte barrier) and improves skin barrier function.

[0069] In one aspect of the present invention, the skin care effect is selected from the group consisting of strengthening the skin barrier, improving the skin barrier function, moisturizing the skin (protecting the skin from dehydration by maintaining, restoring, and / or improving skin moisture), promoting skin repair, and any combination thereof.

[0070] In one embodiment, a skincare composition for providing at least one skincare effect to a subject requiring it comprises an effective amount of Bacillus velezensis ferment extract, the Bacillus velezensis The effective amount of the velezensis fermented extract is, on a weight basis with respect to the total weight of the skincare composition, 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%, and 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%,67%,6 8%, 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%, and a maximum of 100%.

[0071] In one embodiment, a skincare composition for providing at least one skincare effect to a subject requiring it comprises an effective amount of Bacillus velezensis ferment extract, the Bacillus velezensis The effective amount of the velezensis fermented extract is, on a volume basis with respect to the total volume of the skincare composition, 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%, and 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%,67%,6 8%, 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%, and a maximum of 100%.

[0072] In one embodiment, the skincare composition is a skincare composition for providing at least one skincare effect to a subject in need thereof, comprising an effective amount of Bacillus velezensis ferment extract or fraction thereof, wherein this at least one skincare effect is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof.

[0073] In one embodiment, the skincare composition is a skincare composition for providing at least one skincare effect in a subject requiring it, comprising an effective amount of Bacillus velezensis ferment extract or fraction thereof, wherein the at least one skincare effect is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof, and the strengthening of the skin barrier and / or improvement of skin barrier function and / or moisturizing the skin and / or promoting skin repair comprises administering the composition to the skin or scalp of the subject to strengthen the skin barrier and / or improve skin barrier function and / or promote skin repair.

[0074] In one embodiment, the composition promotes skin repair through the production of flagellin.

[0075] In one embodiment, the skincare composition is a skincare composition for providing at least one skincare effect to a subject in need thereof, the composition comprising an effective amount of Bacillus velezensis ferment extract or fraction thereof, the at least one skincare effect being selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof, the composition comprising an effective amount of Bacillus velezensis Fermented extract of *Velezensis* is added by weight to the total weight of the composition in the following amounts: approximately 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%, and 1%. Contains 6%, 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 100% by weight.

[0076] In one embodiment, the skincare composition is a skincare composition for providing at least one skincare effect to a subject in need thereof, the composition comprising an effective amount of Bacillus velezensis ferment extract or fraction thereof, the at least one skincare effect being selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof, the composition comprising an effective amount of Bacillus velezensis The velezensis fermentation extract is added to the total volume of the composition in amounts of approximately 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%, and 15%, respectively, on a volume basis. Includes 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 100%.

[0077] In one embodiment, the skincare composition is a composition for providing at least one skincare effect to a target that requires it, the composition comprising an effective amount of Bacillus velezensis ferment extract or a fraction thereof, the at least one skincare effect being selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof, the Bacillus velezensis ferment extract being selected from the group consisting of cell-free supernatant obtained from Bacillus velezensis ferment, whole broth ferment extract obtained from Bacillus velezensis ferment, and any one combination thereof.

[0078] In one embodiment, the skincare composition is a composition for providing at least one skincare effect as described herein, wherein the Bacillus velezensis fermentation extract is selected from the group consisting of cell-free supernatant obtained from Bacillus velezensis ferment, cell pellet extract obtained from Bacillus velezensis ferment, whole broth fermentation extract obtained from Bacillus velezensis ferment, and any one combination thereof.

[0079] In one embodiment, the skincare composition is a composition for providing at least one skincare effect as described herein, and the Bacillus velezensis fermentation extract or fraction thereof is provided by Westerdijk Fungal Biodiversity under the number CBS147469. Westerdijk Fungal Biodiversity has a B. velezensis strain, numbered CBS151099, with a 16S ribosomal RNA sequence exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the World Federation of Biodiversities Institute (WFDB). For the 16S ribosomal RNA sequence of B. velezensis E04_spo deposited at the Institute (WFDB), 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%, and 99.9% were obtained. It is obtained from a fermented product of B. velezensis selected from the group consisting of B. velezensis strains having 16S ribosomal RNA sequences exhibiting sequence similarity of 2%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100%, and any one combination thereof.

[0080] In one embodiment, the skincare composition is a skincare composition for providing at least one skincare effect in a subject requiring it, the composition comprising an effective amount of Bacillus velezensis ferment extract or fraction thereof, the at least one skincare effect being selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any combination thereof, wherein the strengthening of the skin barrier, and / or the improvement of skin barrier function, and / or the moisturizing of the skin occurs through an increase in tight junctions, an increase in desmosomes, an increase in cell-cell adhesion, an increase in keratinization, and any combination thereof.

[0081] In one embodiment, the increase in tight junctions occurs through increased expression of genes 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 combination thereof, compared to the expression of the genes caused by a control composition that is identical to the skincare composition except for the absence of the effective amount of the Bacillus velezensis fermentation extract or fraction thereof.

[0082] In one embodiment, the increase in desmosomes occurs through increased expression of genes 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 combination thereof, compared to the expression of the genes caused by a control composition that is identical to the skincare composition except that it does not contain the effective amount of the Bacillus velezensis fermentation extract or fraction thereof.

[0083] In one embodiment, the increase in cell-cell adhesion is due to an effective amount of the Bacillus berezensis 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 combination thereof. This occurs through increased gene expression compared to the expression of the gene caused by a control composition which is identical to the skincare composition except for the absence of the velezensis fermentation extract or fraction thereof.

[0084] In one embodiment, the increase in keratinization occurs through increased expression of genes 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 combination thereof, compared to the expression of the genes caused by a control composition that is identical to the skincare composition except that it does not contain the effective amount of the Bacillus velezensis fermentation extract or fraction thereof.

[0085] In one embodiment, the skincare composition described herein further comprises additional compounds selected from the group consisting of fragrances, excipients, preservatives, pH adjusters, aqueous carriers, alcohol carriers, emollients, coagulants, hydrates, emulsifiers, solubilizers, surfactants, thickeners, salts, and any combination thereof.

[0086] Examples of preservatives include, but are not limited to, parabens, sodium benzoate, potassium sorbate, phenylethyl alcohol, ethyl lauryl alginate (LAE), and any combination thereof.

[0087] Examples of pH adjusting agents include, but are not limited to, weak acids, strong acids, any compound capable of adjusting pH, such as citric acid (but not limited to), or any combination thereof.

[0088] In one embodiment, the composition is a ferment of a Bacillus velezensis strain variant, a fermented extract of a Bacillus velezensis strain variant, or a fraction of the ferment of the Bacillus velezensis strain variant, wherein the Bacillus velezensis strain variant is obtained by knocking out the spore-forming gene of the parent B. velezensis strain. Obtained from B. velezensis, the variant ferment, the variant ferment extract, or the fraction of the variant ferment exhibits increased activity compared to the activity of the ferment, ferment extract, or fraction derived from the parent B. velezensis strain, and the activity is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any one of these combinations.

[0089] In one embodiment, the composition is a ferment of a Bacillus velezensis strain variant, a fermented extract of a Bacillus velezensis strain variant, or a fraction of the ferment of the B. velezensis strain variant. The velezensis strain variant is obtained from the parent B. velezensis strain by knocking out the spore-forming gene of the parent B. velezensis strain. The variant ferment, the variant ferment extract, or the fraction of the variant ferment exhibits increased activity compared to the activity of the ferment, ferment extract, or fraction derived from the parent B. velezensis strain. This activity is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any one of these combinations, and the spore-forming gene is spoIIE.

[0090] In one embodiment, the composition is a ferment of a Bacillus velezensis strain variant, a fermented extract of a Bacillus velezensis strain variant, or a fraction of the ferment of the B. velezensis strain variant. The velezensis) strain variant is obtained from the parent B. velezensis strain by knocking out the spore-forming gene of the parent B. velezensis strain, and the variant ferment, the variant ferment extract, or the fraction of the variant ferment exhibits increased activity compared to the activity of the ferment, ferment extract, or fraction derived from the parent B. velezensis strain, and the increased activity is selected from the group consisting of skin barrier strengthening, improvement of skin barrier function, skin moisturizing, and any combination thereof, and the skin barrier strengthening and / or improvement of skin barrier function and / or skin moisturizing occurs through an increase in tight junctions, an increase in desmosomes, an increase in cell-cell adhesion, an increase in keratinization, and any combination thereof.

[0091] Any effective amount of the skincare composition described herein or said skincare composition may be formulated in a skincare product.

[0092] As used herein, “skincare product” means a product or formulation comprising a skincare composition as described herein, and includes, but is not limited to, cosmetics, aqueous solutions, emulsions, serums, jellies, patches, lotions, topical moisturizers, creams, pastes, balms, ointments, pomades, gels, liquids, sprays, foams, kits, or any one of these.

[0093] In one embodiment, the skincare product is a skincare product comprising the skincare composition described herein.

[0094] In one embodiment, the skincare product is a skincare product comprising the skincare composition described herein and one or more dermatologically or cosmetically acceptable ingredients.

[0095] In one embodiment, the skincare product is a skincare product comprising a skincare composition for providing at least one skincare effect to a target that requires it, the composition comprising an effective amount of Bacillus velezensis ferment extract or fraction thereof.

[0096] In one embodiment, the skincare product comprises one or more dermatologically or skincare-acceptable ingredients and a skincare composition for providing at least one skincare effect in a subject requiring it, the composition comprising an effective amount of B. velezensis ferment extract or fraction thereof, wherein the at least one skincare effect is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof.

[0097] In one embodiment, the skincare product comprises a skincare composition for providing at least one skincare effect to a subject in need thereof, the composition being manufactured by Westerdijk Fungal Biodiversity under the number CBS147469. Westerdijk Fungal Biodiversity has a B. velezensis strain, numbered CBS151099, with a 16S ribosomal RNA sequence exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the World Federation of Biodiversities Institute (WFDB). For the 16S ribosomal RNA sequence of B. velezensis E04_spo deposited at the Institute (WFDB), 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%, and 99.93% were obtained. This product contains a Bacillus velezensis fermentation extract or fraction thereof, obtained from a Bacillus velezensis fermentation product selected from the group consisting of B. velezensis strains having 16S ribosomal RNA sequences exhibiting 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, and any combination thereof.

[0098] In one embodiment, the skincare product is a skincare product comprising, by weight, at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% of the skincare composition described herein, based on the total weight of the skincare product.

[0099] 11b. In one embodiment, the skincare product is a skincare composition comprising, by volume, at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% of the skincare composition described herein, based on the total volume of the skincare product.

[0100] In one embodiment, the skincare product is formulated for topical administration. In another embodiment, the skincare product is formulated for topical administration to the skin or scalp.

[0101] In one embodiment, the skincare product is a skincare product according to this specification, selected from the group consisting of lotions, serums, jellies, creams, gels, hydrogels, emulsions, solid cosmetics, masks, patches, shampoos, loose powders or compact powders, liquid suspensions or solutions, spray solutions, and sticks.

[0102] Topical formulations for use in the present invention may be in any form suitable for application to the scalp or skin surface, such as creams, lotions, sprays, solutions, gels, ointments, pastes, plasters, cosmetics, bioadhesives, or suspensions, and / or may be prepared to contain liposomes, micelles, and / or microspheres. Such formulations may be used in combination with a sealing cover to retain moisture within the formulation when the formulation is applied to the body surface and thereafter when moisture evaporates from the body surface.

[0103] Topical formulations include those in which the active ingredient is 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). The components of this type of vehicle may include water, an aqueous buffer, a non-aqueous solvent (e.g., ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycoflor, or glycerol), and an oil (e.g., mineral oil such as liquid paraffin, natural or synthetic triglycerides, or silicone oil such as dimethicone). In particular, depending on the properties of the formulation as well as its intended use and application site, the dermatological vehicle used may contain one or more components selected from the following list (for example, components other than water if the formulation is an aqueous gel): solubilizers or solvents (e.g., β-cyclodextrin, e.g., hydroxypropyl β-cyclodextrin, or alcohols or polyols, e.g., ethanol, propylene glycol, or glycerol), thickeners (e.g., hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, or carbomer), gelling agents (e.g., polyoxyethylene-polyoxypropylene copolymer), preservatives (e.g., benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorbutol, benzoate, potassium sorbate, or EDTA or its salts), and pH buffers (e.g., a mixture of dihydrogen phosphate and hydrogen phosphate, or a mixture of citric acid and hydrogen phosphate).

[0104] Examples of skincare products include liquid lotions (true solutions) containing water as a solvent and water-soluble additives (solutes), such as surfactants, fragrances, colorants, preservatives, pH adjusters, chelating agents, or any combination thereof.

[0105] Examples of skincare products include dispersions such as emulsions (but are not limited to: liquid-in-liquid [water-in-oil W / O, O / W, W / O / W], suspensions [solid / liquid or liquid / solid], aerosols [liquid / gas or solid / gas], foams / mousses [gas / liquid or gas / emulsion or gas / solid]). Examples of oil-in-water [O / W] emulsions include, but are not limited to, a combination of an aqueous phase, an emulsifier, a fatty phase, and at least one additive. The aqueous phase may contain water, a wetting agent, and a stabilizer (synthetic polymers, carbomers, natural polymers, xanthan gum, acacia gum, carrageenan, gellan, or any combination thereof, but are not limited). Examples of emulsifiers include, but are not limited to, anionic emulsifiers, cationic emulsifiers, nonionic emulsifiers, amphoteric emulsifiers, silicone emulsifiers, and self-emulsifiers. Examples of fatty phase components (lipophilic components) include, but are not limited to, waxes, butters, fatty acid esters, triglycerides, vegetable oils, mineral oils (paraffin), silicones, and thickeners / oil gelling agents. Examples of additives include, but are not limited to, preservatives, fragrances (mostly lipophilic), colorants, antioxidants, chelating agents, surfactants, pH adjusters (citric acid, lactic acid, AHA), neutralizing agents / strong bases such as NaOH, trimethylamine (for gelling acrylic polymers), and powders.

[0106] Examples of skincare products include aqueous gels containing an aqueous phase (water, humectants, and surfactants), gelling agents (but not limited to synthetic polymers, natural polymers, xanthan gum, acacia gum, carrageenan, gellan, etc.), and additives (but not limited to fragrances, high-HLB surfactants, colorants, surfactants, preservatives, pH adjusters, neutralizing agents, powders, etc.).

[0107] Examples of skincare products include cleansing agents / surfactants (such as shampoos, shower gels, micellar waters, etc.) that contain an aqueous phase (water, humectants), surfactants, additives (but not limited to fragrances, high-HLB surfactants, colorants, surfactants, preservatives, pH adjusters, neutralizing agents, powders, etc.) and optionally gelling agents (but not limited to synthetic polymers, natural polymers, xanthan gum, acacia gum, carrageenan, gellan, etc.).

[0108] The skincare products (formulations) of the present invention may also incorporate dermatologically or skincare-acceptable carriers, which may be any carrier used in the art. Examples 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 aqueous and emulsion mixtures of such carriers.

[0109] As used herein, the terms “dermatologically acceptable,” “dermatologically acceptable carrier,” “skin care acceptable,” or “skin care acceptable carrier” refer to a compound or composition that can be incorporated into a dermatological or skin care formulation without causing undesirable biological effects or undesirable interactions with other components of the formulation.

[0110] As used herein, “carrier” or “vehicle” refers to a carrier material suitable for incorporation into a topically applied composition. Useful carriers and vehicles herein include any material known in the art that is non-toxic and does not interact in a detrimental manner with other components of the formulation containing it.

[0111] The term "aqueous" refers to a formulation that contains water or becomes water-containing after being applied to skin or mucous membrane tissue.

[0112] The skin care products described herein may further contain one or more dermatologically or skin-skin-acceptable known components or other components effective for use in skin care, provided that the optional components are physically and chemically compatible with the essential components described herein, or otherwise do not unduly impair the stability, aesthetics, or performance of the product. Non-exclusive examples of such optional components are disclosed in the International Skin Care Ingredient Dictionary, Ninth Edition, 2002 and the CTFA Skin Care Ingredient Handbook, Tenth Edition, 2004.

[0113] In one embodiment, the dermatologically or skin-skin-acceptable components are dermatologically acceptable carriers comprising about 10% to about 99.9% by weight, or instead about 50% to about 95% by weight, or instead about 75% to about 95% by weight. Suitable carriers for use with the composition include, for example, those used in the formulation of mousses, tonics, gels, and skin moisturizing lotions. The carriers include water, organic oils, silicones, such as volatile silicones, amino or non-aminosilicone gums or oils and mixtures thereof, mineral oils, vegetable oils, such as olive oil, castor oil, rapeseed oil, coconut oil, wheat germ oil, sweet peach oil, avocado oil, macadamia oil, apricot oil, safflower oil, kukui oil, cinnamon oil, malva seed oil, lemon oil and mixtures thereof, waxes, and organic compounds, such as C2-C2 10 Alkanes, acetone, methyl ethyl ketone, volatile organic compounds C1-C1 12 Alcohol, C1~C 20 Acids and esters of C1-C8 alcohols, such as methyl acetate, butyl acetate, ethyl acetate, and isopropyl myristate, dimethoxyethane, diethoxyethane, C 10 ~C 30 Fatty alcohols, such as lauryl alcohol, cetyl alcohol, stearyl alcohol and behenyl alcohol, C 10 ~C 30Fatty acids, such as lauric acid and stearic acid, C 10 ~C 30 Fatty amides, such as lauric acid diethanolamide, C 10 ~C 30 Fatty alkyl esters, such as C 10 ~C 30 It may include fatty alkyl benzoate, hydroxypropyl cellulose and mixtures thereof. In one embodiment, the carrier includes water, fatty alcohols, volatile organic alcohols and mixtures thereof. Those skilled in the art can formulate other carriers.

[0114] The skin care products described herein may further include a gelling agent that promotes imparting a desired viscosity to the composition, from about 0.1% to about 10%, alternatively from about 0.2% to about 5.0%. Non-limiting examples of suitable optional gelling agents include crosslinked carboxylic acid polymers, non-neutralized crosslinked carboxylic acid polymers, non-neutralized modified crosslinked carboxylic acid polymers, crosslinked ethylene / maleic anhydride copolymers, non-neutralized crosslinked ethylene / maleic anhydride copolymers (e.g., EMA81 commercially available from Monsanto), non-neutralized crosslinked alkyl ether / acrylic acid copolymers (e.g., SALCARE™ SC90 commercially available from Allied Colloids), sodium polyacrylate, non-neutralized crosslinked copolymers of mineral oil and PEG-1 trideceth-6 (e.g., SALCARE™ SC91 commercially available from Allied Colloids), non-neutralized crosslinked copolymers of methyl vinyl ether and maleic anhydride (e.g., STABILEZE™ QM-PVM / MA copolymer commercially available from International Specialty Products), hydrophobically modified nonionic cellulose polymers, hydrophobically modified ethoxylated urethane polymers (e.g., UCARE™ Polyphobe Series of alkali swellable polymers commercially available from Union Carbide) and combinations thereof. In this regard, the term "non-neutralized" means that the optional polymer and copolymer gelling agent materials contain non-neutralized acid monomers.

[0115] A dermatologically or skin-skin-acceptable medium may contain fatty substances in a proportion generally of about 10 to about 90% by weight of the total weight of the product, and the fatty phase may contain at least one liquid, solid, or semi-solid fatty substance. Fatty substances include, but are not limited to, oils, waxes, gum-like substances, and so-called paste-like fatty substances. Alternatively, the product may be in the form of a stable dispersion such as a water-in-oil or oil-in-water emulsion. In addition, the skin-skin product may contain one or more conventional skin-skin or dermatological additives or auxiliaries, such as, but are not limited to, antioxidants, preservatives, fillers, surfactants, UVA and / or UVB sunscreens, fragrances, thickeners, humectants, and anionic, nonionic, or amphoteric polymers, as well as dyes or pigments (colorants).

[0116] A dermatologically acceptable carrier may be a moisturizing formulation containing at least one emulsifier, at least one surfactant, or any combination thereof.

[0117] Skincare compositions and skincare products may further contain materials for active skincare ingredients, including sunscreens, moisturizers, humectants, skin-beneficial substances, adhesion-enhancing agents such as surfactants, occlusive agents, moisture barriers, lubricants, emollients, anti-aging agents, antistatic agents, abrasives, antimicrobial agents, conditioners, scrubs, fragrances, viscosity enhancers, salts, lipids, phospholipids, vitamins, foam stabilizers, pH adjusters, preservatives, suspending agents, silicone oils, silicone derivatives, essential oils, oils, fats, fatty acids, fatty acid esters, fatty alcohols, waxes, polyols, hydrocarbons, and mixtures thereof.

[0118] Other ingredients that may be included in a skincare composition or skincare product include at least one active ingredient that provides a skincare effect or a moisturizing effect on the skin for treating or preventing skin diseases, 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 thereof, and one or more natural moisturizing factors (such as ceramide, hyaluronic acid, glycerin, squalane, amino acids, cholesterol). Examples of but not limited to fatty acids, triglycerides, phospholipids, sphingoglycolipids, urea, linoleic acid, glycosaminoglycans, mucopolysaccharides, sodium lactate or sodium pyrrolidone carboxylate, etc., 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 peach oil, sunflower oil, tea tree oil, shea butter, coconut oil, cholesterol, cholesterol esters, wax esters, fatty acids, and orange oil are also included.

[0119] Any number of dermatologically acceptable ingredients commonly used in skincare products, such as skin conditioning agents and skin colorants, can also be incorporated into this skincare product.

[0120] Skin conditioning agents as defined herein include, but are not limited to, astringents that tighten the skin, scrubs that remove dead skin cells, emollients that promote the maintenance of a smooth, supple, and flexible appearance, humectants that increase the moisture content of the skin surface, occlusive agents that slow the evaporation of water from the skin surface, and various compounds that enhance the appearance of dry or damaged skin, reduce peeling, and restore suppleness. Skin conditioning agents are well known in the art (see, for example, Green et al. (International Publication No. 01 / 07009)) and are commercially available from various suppliers. Suitable examples of skin conditioning agents include, but are not limited to, lactobionic acid, gluconic acid, alpha-hydroxy acid, beta-hydroxy acid, polyol, hyaluronic acid, D,L-panthenol, polysalicylate, vitamin A palmitate, vitamin E acetate, glycerin, sorbitol, silicone, silicone derivatives, lanolin, natural oils, xylitol, fucose, rhamnose, xylitol, betaine, and triglyceride esters. As skin conditioning agents, polysalicylate, propylene glycol (CAS No. 57-55-6, Dow Chemical, Midland, MI), glycerin (CAS No. 56-81-5, Proctor & Gamble Co., Cincinnati, OH), glycolic acid (CAS No. 79-14-1, DuPont Co., Wilmington, DE), 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 Examples include 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).Polysalicylic acid can be prepared by the method described in U.S. Patent No. 4,855,483 by White et al., which is incorporated herein by reference. Glucaric acid can be synthesized using the method described by Merbouh et al. (Carbohydr. Res. 336:75-78 (2001)). 3-Hydroxyvaleric acid can be prepared as described in International Publication No. 02 / 012530 by Bramucci.

[0121] Skincare compositions and skincare products may include, but are not limited to, skincare additives such as colorants / dyes, fragrances, surfactants, preservatives, pH adjusters, chelating agents, and antioxidants.

[0122] The skincare compositions and skincare products described herein may be part of a kit for providing one or more skincare effects, such as a kit for providing at least one skincare effect as described herein, but are not limited to such kits.

[0123] In one embodiment, the kit comprises an effective amount of Bacillus velezensis ferment, Bacillus velezensis ferment extract and / or fraction thereof for providing at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin (protecting the skin from dehydration by maintaining, restoring, and / or improving skin moisture), promoting skin repair, and any combination thereof.

[0124] A way to provide at least one skincare benefit The skincare compositions and skincare products described herein may be used in a manner that provides at least one skincare effect. In one embodiment, the at least one skincare effect is selected from the group consisting of strengthening the skin barrier, improving the skin barrier function, moisturizing the skin (protecting the skin from dehydration by maintaining, restoring and / or strengthening skin moisture), promoting skin repair, and any one combination thereof.

[0125] In one embodiment, the method is a method for providing at least one skincare effect to a subject in need thereof, and the method includes topically administering a skincare composition or skincare product described herein to the skin or scalp of the subject.

[0126] In one embodiment, the method is a method for providing at least one skincare effect to a subject in need thereof, the method comprising topically administering a skincare composition or a skincare product containing the composition to the skin of the subject, the composition comprising an effective amount of B. velezensis ferment extract or a fraction thereof, and the at least one skincare effect is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof.

[0127] In one embodiment, the method provides at least one skincare effect to a subject in need, the method comprising topically administering a composition containing an effective amount of B. velezensis ferment extract or a fraction thereof to the subject in need, the at least one skincare effect being selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof.

[0128] In one embodiment, the method provides a subject in need of at least one skin care effect, comprising topically administering a composition containing an effective amount of B. velezensis ferment extract or fraction thereof to the subject in need, wherein the at least one skin care effect is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof, and the Bacillus velezensis ferment extract or fraction thereof is distributed by Westerdijk Fungal Biodiversity under the number CBS147469. Westerdijk Fungal Biodiversity has a B. velezensis strain, numbered CBS151099, with a 16S ribosomal RNA sequence exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the World Federation of Biodiversities Institute (WFDB). For the 16S ribosomal RNA sequence of B. velezensis E04_spo deposited at the Institute (WFDB), 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%, and 99.9% were obtained. It is obtained from a fermented product of B. velezensis selected from the group consisting of B. velezensis strains having 16S ribosomal RNA sequences exhibiting sequence similarity of 2%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100%, and any one combination thereof.

[0129] In one embodiment, the method is a method for strengthening the skin barrier in a subject, and the method comprises topically administering a skincare composition or skincare product described in any of the preceding claims to the skin or scalp of the subject.

[0130] In one embodiment, the method is a method for strengthening the skin barrier in a subject, and the method comprises topically administering a skincare composition or skincare product containing an effective amount of B. velezensis fermented extract or a fraction thereof to the skin or scalp of the subject.

[0131] In one embodiment, the method is a method for improving the skin barrier function in a subject, and the method comprises topically administering a skincare composition or skincare product described in any of the preceding claims to the skin or scalp of the subject.

[0132] In one embodiment, the method is a method for improving the skin barrier function in a subject, and the method comprises topically administering a skincare composition or skincare product containing an effective amount of B. velezensis fermented extract or a fraction thereof to the skin or scalp of the subject.

[0133] In one embodiment, the method is a method for moisturizing the skin of a subject, which includes topically administering a skincare composition or skincare product described in any of the preceding claims to the skin or scalp of the subject.

[0134] In one embodiment, the method is a method for moisturizing the skin of a subject, which includes topically administering a skincare composition containing an effective amount of B. velezensis fermented extract or a fraction thereof to the skin or scalp of the subject.

[0135] In one embodiment, the skincare product is administered to a subject in need thereof, and the skincare product is present in an amount of at least approximately 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%, and 8% by weight relative to the total weight of the skincare product. Contains %, 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%, and up to 50% of the skincare compositions described herein.

[0136] In one embodiment, the skincare product is administered to a subject in need, and the skincare product is present in a volume-based amount of at least approximately 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%, and 8% of the total volume of the skincare product. Contains %, 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%, and up to 50% of the skincare compositions described herein.

[0137] A method for increasing the activity of a fermented extract of Bacillus velezensis strain and its composition. A method for increasing the activity of a fermented extract of the Bacillus velezensis strain to provide at least one skincare effect is disclosed herein.

[0138] The inventors have unexpectedly observed that when a spore-forming gene, such as SpoIIE, is knocked out in a parent B.velezensis strain (e.g., B.velezensis E04, but not limited) to generate a B.velezensis variant strain (e.g., B.velezensis E04_spo, but not limited), the activity of the ferment, ferment extract, or fraction derived from the B.velezensis variant strain increases compared to the activity of the ferment, ferment extract, or fraction thereof derived from the parent B.velezensis strain. This activity is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any one of these combinations.

[0139] In one embodiment, the present method is a method for increasing the activity of a fermented extract of a Bacillus velezensis strain to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject that requires it, the method comprising: (a) providing a parent Bacillus velezensis strain capable of producing an effective amount of fermented extract to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject that requires it; (b) modifying the parent Bacillus velezensis strain by knocking out at least one spore-forming gene from the genome of the Bacillus velezensis strain, thereby generating a variant strain that cannot form spores; and (c) the Bacillus velezensis strain The present invention comprises producing a fermented extract of a Bacillus velezensis variant strain, wherein the fermented extract of the Bacillus velezensis variant strain exhibits increased activity compared to the fermented extract of the parent Bacillus velezensis strain, providing at least one skincare effect selected from the group consisting of skin barrier strengthening, improvement of skin barrier function, skin moisturizing, and any combination thereof, in subjects requiring such effects.

[0140] In one embodiment, the method is a method for increasing the activity of a fermented extract of a Bacillus velezensis strain to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject that needs it, the method being (a) to provide a parent Bacillus velezensis strain capable of producing an effective amount of fermented extract to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject that needs it, and (b) to knock out at least one spore-forming gene from the genome of the Bacillus velezensis strain, thereby increasing the activity of the parent Bacillus velezensis strain (c) modifying the Bacillus velezensis strain to produce a variant strain that is unable to form spores, wherein at least one spore-forming gene is SpoIIE; and (c) producing a fermented extract of the Bacillus velezensis variant strain, wherein the fermented extract of the Bacillus velezensis variant strain has increased activity compared to the activity of the fermented extract of the parent Bacillus velezensis strain to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to subjects in need.

[0141] General definition All patent and non-patent literature disclosures cited herein are incorporated herein by reference in their entirety.

[0142] This disclosure uses numerous terms and abbreviations. Unless otherwise specified, the following definitions apply.

[0143] As used herein, the articles “a,” “an,” and “the” preceding an element or component of the present invention are intended to be non-limiting in terms of the number of occurrences of that element or component. Therefore, “a,” “an,” and “the” should be read as including one or at least one, and the singular form of an element or component also includes the plural form unless it specifically implies that its number is singular.

[0144] Where quantities, concentrations, or other values ​​or parameters are given as a range, a preferred range, or a list of preferred higher and preferred lower values, this should be understood as specifically disclosing any range formed by any pair of upper or preferred higher values ​​of any range and lower or preferred lower values ​​of any range, regardless of whether the ranges are disclosed separately. Where a range of numerical values ​​is enumerated herein, unless otherwise specified, that range is intended to include its endpoint and all integers and fractions within that range. The ranges of the present invention are not intended to be limited to any specific values ​​enumerated when defining a range.

[0145] The use of numerical values ​​within the various ranges specified herein is described as approximations, as if preceded by the term “approximately” before either the minimum or maximum value within the specified range, unless otherwise explicitly indicated. In this form, a small variation above or below the specified range can be used to achieve substantially the same results as values ​​within that range. The disclosure of these ranges is also intended to include a continuous range encompassing each and every value between the minimum and maximum values. As used herein, the term “approximately” modifying the amount of components or reactants used means, for example, through typical weighing and liquid handling procedures used in the real world to produce concentrates or solutions of use, through accidental errors in these procedures, through differences in the manufacture, source, or purity of components used to create a composition or to carry out the method. The term “approximately” also includes different amounts resulting from different equilibrium conditions for the resulting composition from a particular initial mixture. Whether modified by the term “approximately” or not, the claims include quantitative equivalents.

[0146] As used herein, the terms “administer” or “to administer” mean the act of introducing one or more microorganisms (microbial strains), ferments, fermentation extracts or fractions thereof, skincare compositions, skincare formulations and / or skincare products to a subject in order to provide at least one skincare effect.

[0147] The administration of one or more microorganisms (microbial strains), fermentates, fermentation extracts or fractions thereof, skincare compositions, skincare formulations and / or skincare products to a subject includes applying or introducing one or more microorganisms (microbial strains), fermentates, fermentation extracts or fractions thereof, skincare compositions, skincare formulations and / or skincare products to the scalp, skin surface and skin cells in vitro or in vivo.

[0148] As used herein, the term “biological contaminants” refers to one or more undesirable and / or pathogenic biological entities, including but not limited to microorganisms, spores, viruses, prions, and mixtures thereof.

[0149] As used herein, the term “including” means the presence of a specific feature, integer, stage, or component referred to in the claims, but does not exclude the presence or addition of one or more other features, integers, stages, components, or groups thereof. The term “including” includes embodiments encompassed by the terms “essentially consisting of” and “consisting of.” Similarly, the term “essentially consisting of” is intended to include embodiments encompassed by the term “consisting of.”

[0150] As used herein, the terms “embodiments” or “disclosures” are not intended to be limiting, but rather apply generally to either the embodiments defined in the claims or those described herein. These terms are used interchangeably herein.

[0151] As used herein, the term “excipient” refers to an inert substance used in a formulation as a carrier for the active ingredient. Excipients may be used to stabilize the active ingredient in the formulation, such as for storage stability. Excipients may also be used to increase the volume of a formulation containing the active ingredient. “Active ingredient” includes substances beneficial for skin care as described herein.

[0152] As used herein, the term “effective amount” means an amount sufficient to obtain the desired effect. The desired effect includes strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any combination thereof.

[0153] As used herein, “prevent,” “prevention,” “prevention,” and their grammatical variations mean a method of partially or completely delaying or preventing the onset or recurrence of one or more of a condition and / or symptoms associated therewith, or a method of preventing an object from acquiring or regaining a condition, or a method of reducing the risk to an object from acquiring or regaining one or more of a condition and / or symptoms associated therewith.

[0154] As used herein, the terms “reduce,” “reduce,” and their grammatical variations relating to a particular feature, characteristic, property, biological process, or phenomenon mean a reduction of that particular feature, characteristic, property, biological process, or phenomenon. This feature, characteristic, property, biological process, or phenomenon may be reduced by more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or more than 100%.

[0155] The terms "weight %", "weight percent (wt.%)", and "weight-weight percent (%w / w)" are used interchangeably herein. Weight percent refers to the percentage of a material by mass contained in a composition, mixture, solution, or product.

[0156] The term "16S rRNA" or "16S ribosomal RNA" refers to the rRNA that constitutes the small subunit of the ribosome in prokaryotes. In bacteria, this sequence can be used to identify and characterize operational taxa.

[0157] The term "ITS" or "internal transcription spacer" refers to regions within ribosome transcripts that are cleaved and degraded during maturation. These sequences can be used for phylogenetic analysis and / or identification of fungi or yeasts.

[0158] The terms humectant, lotion, or body lotion refer to low-to-medium viscosity emulsions of oil and water, most of which are oil-in-water, although some are water-in-oil. Their primary effect in skincare applications is to hydrate the skin or reduce skin water loss. Almost all humectants contain a combination of emollients, occlusive agents, and wetting agents. Moisturizing agents are primarily lipids and oils, which hydrate and improve the appearance of the skin. A wide variety of suitable emollients are known and can be used herein (the 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, DC, 7th Edition, 1997) (referred to as the "ICI Handbook") contains many examples of suitable materials). Occlusive agents such as petrolatum, lanolin, and beeswax reduce transepidermal water loss by creating a hydrophobic barrier on the skin. Humectants such as glycerol and urea can attract water from the external environment and enhance water absorption from the dermis to the epidermis. In addition, moisturizing formulations may contain emulsifiers to maintain emulsion stability and thickeners may be used to achieve the desired viscosity and skin feel. A wide variety of other ingredients such as fragrances, dyes, preservatives, therapeutic agents, proteins, and stabilizers are commonly added to impart attributes preferred by other consumers.

[0159] The terms “percent (%) sequence identity” or “percent (%) sequence similarity,” as used herein in relation to a reference sequence, are defined as the percentage of nucleotide residues in a candidate sequence that are identical to residues in a reference polynucleotide sequence after the sequence has been optimally aligned by inserting gaps as necessary to maximize sequence identity %.

[0160] As used herein, the terms “probiotics” or “probiotic microorganisms” are interchangeable herein and refer to living microorganisms (including, for example, bacteria or yeasts) that, when administered in sufficient quantities (topically or orally), have a beneficial effect on a host organism, i.e., by providing one or more demonstrable benefits to the host organism. There are no lower or upper limits on the use of probiotics, but at least 10 per day is recommended. 6 ~10 12 Preferably at least 10 6 ~10 10 Preferably 10 8 ~10 9 It has been suggested that CFU is effective in obtaining beneficial effects in the target area.

[0161] As used herein, a microbial "strain" refers to a microorganism (such as a bacterium or fungus) that remains genetically unchanged even as it grows or multiplies. This encompasses diversity within the same microorganism.

[0162] As used herein, the term “biologically pure strain” means a strain that does not contain other microbial strains in amounts sufficient to prevent strain replication or to be detected by conventional techniques. “Isolated,” as used herein in reference to the organisms and cultures described herein, includes not only biologically pure strains but also any culture of an organism that is grown or maintained in a manner different from that found in nature.

[0163] In one embodiment, the skin cells described herein are mammalian skin cells such as human skin cells or animal skin cells.

[0164] The terms “sequence identity” or “sequence similarity,” as used herein, mean that two polynucleotide sequences (i.e., a candidate sequence and a reference sequence) are identical (i.e., 100% sequence identity) or similar (i.e., similar on a nucleotide-to-nucleotide basis) over the entire length of the candidate sequence. When the candidate sequence and the reference sequence are optimally aligned, the candidate sequence may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions). Optimal alignment of sequences for determining sequence identity can be performed using any number of publicly available local alignment algorithms known in the Art (e.g., ALIGN or Megalign (DNASTAR)), or by scrutiny.

[0165] Throughout this specification, all numerical upper limits are intended to include all lower numerical limits as if such lower numerical limits were explicitly stated herein. Throughout this specification, all numerical lower limits will include all higher numerical limits as if such higher numerical limits were explicitly stated herein. Throughout this specification, all numerical ranges will include all narrower numerical ranges that fall within such wider numerical ranges as if all such narrower numerical ranges were explicitly stated herein.

[0166] Unless otherwise defined herein, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention pertains.

[0167] Non-limiting examples of compositions and methods disclosed herein include: 1. A skincare composition for providing at least one skincare effect in a subject requiring it, comprising an effective amount of Bacillus velezensis ferment extract or a fraction thereof, wherein the at least one skincare effect is selected from the group consisting of strengthening the skin barrier, improving the skin barrier function, moisturizing the skin (protecting the skin from dehydration by maintaining, restoring, and / or improving skin moisture), promoting skin repair, and any one combination thereof. 1a. A skincare composition for providing at least one skincare effect in a subject requiring it, comprising an effective amount of Bacillus velezensis ferment extract or fraction thereof, wherein the at least one skincare effect is selected from the group consisting of strengthening the skin barrier, improving the skin barrier function, moisturizing the skin (protecting the skin from dehydration by maintaining, restoring, and / or improving skin moisture), promoting skin repair, and any one combination thereof. 1b. A skincare composition according to Embodiment 1, wherein the strengthening of the skin barrier comprises administering the composition to the skin or scalp of the target in order to strengthen the skin barrier of the target. 1c. A skincare composition according to Embodiment 1, wherein the improvement of the skin barrier function comprises administering the composition to the skin or scalp of a target in order to improve the skin barrier function of the target. 1d. A skincare composition according to Embodiment 1, wherein the skin moisturizing comprises administering the composition to the target skin or scalp in order to moisturize the target skin or scalp. 1e. A skincare composition according to Embodiment 1, wherein the promotion of skin repair comprises administering the composition to the skin or scalp of a subject in order to promote the skin repair of the subject. 1f. A skincare composition according to Embodiment 1 or 1e, which promotes skin repair through the production of flagellin. 1g. A skincare composition according to any of the preceding embodiments, comprising approximately 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%, 1g of the total weight of the composition. A skincare composition containing an effective amount of Bacillus velezensis ferment extract in the following proportions: 7%, 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 100%. 1h. A skincare composition according to any of the preceding embodiments, wherein the total volume of the composition is approximately 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%, 1 A skincare composition containing an effective amount of Bacillus velezensis ferment extract in the following proportions: 7%, 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 100%. 2. A skincare composition according to Embodiment 1, wherein the Bacillus velezensis fermentation extract is selected from the group consisting of cell-free supernatant obtained from Bacillus velezensis ferment, whole broth fermentation extract obtained from Bacillus velezensis ferment, and any one combination thereof. 2b. A skincare composition according to Embodiment 1, wherein the Bacillus velezensis fermentation extract is selected from the group consisting of cell-free supernatant obtained from Bacillus velezensis ferment, cell pellet extract obtained from Bacillus velezensis ferment, whole broth fermentation extract obtained from Bacillus velezensis ferment, and any one combination thereof. 2c. The skincare composition according to Embodiment 1, wherein the Bacillus velezensis fermentation extract or fraction thereof is provided to Westerdijk Fungal Biodiversity under the number CBS147469. Westerdijk Fungal Biodiversity has a B. velezensis strain, numbered CBS151099, with a 16S ribosomal RNA sequence exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the World Federation of Biodiversities Institute (WFDB). For the 16S ribosomal RNA sequence of B. velezensis E04_spo deposited at the Institute (WFDB), 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%, and 9 A skincare composition obtained from a ferment of B. velezensis selected from the group consisting of B. velezensis strains having 16S ribosomal RNA sequences exhibiting sequence similarity of 9.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100%, and any one combination thereof. 3. A skincare composition according to Embodiment 1, wherein the strengthening of the skin barrier and / or the improvement of the skin barrier function and / or the moisturizing of the skin occurs through an increase in tight junctions, an increase in desmosomes, an increase in cell-cell adhesion, an increase in keratinization and any combination thereof. 4. A skincare composition according to Embodiment 3, wherein the increase in tight junctions occurs through 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 combination thereof, compared to the expression of the gene caused by a control composition which is identical to the skincare composition except that it does not contain an effective amount of the Bacillus velezensis fermentation extract or fraction thereof. 4b. A skincare composition according to Embodiment 3, wherein the increase in tight junctions occurs through 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 combination thereof, compared to the expression of the gene caused by a control composition lacking an effective amount of the Bacillus velezensis fermentation extract or fraction thereof. 5. A skincare composition according to Embodiment 3, wherein the increase in desmosomes occurs through 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 combination thereof, compared to the expression of the gene caused by a control composition which is identical to the skincare composition except that it does not contain an effective amount of the Bacillus velezensis fermentation extract or fraction thereof. 5b. A skincare composition according to Embodiment 3, wherein the increase in desmosomes occurs through 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 combination thereof, compared to the expression of the gene caused by a control composition lacking the effective amount of the Bacillus velezensis fermentation extract or fraction thereof. 6. The skincare composition according to Embodiment 3, wherein the increase in cell-cell adhesion is due to an effective amount of the Bacillus berezensis 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 combination thereof. A skincare composition that results from increased expression of the gene compared to the expression of the gene caused by a control composition which is identical to the skincare composition except that it does not contain a fermented extract or fraction thereof (velezensis). 6b. The skincare composition according to Embodiment 3, wherein the increase in cell-cell adhesion is due to 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 combination thereof, of the Bacillus berezensis gene A skincare composition that results from increased expression of the gene compared to the expression of the gene caused by a control composition lacking the velezensis fermentation extract or fraction thereof. 7. A skincare composition according to Embodiment 3, wherein the increase in keratinization occurs through 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 combination thereof, compared to the expression of the gene caused by a control composition which is identical to the skincare composition except that it does not contain an effective amount of the Bacillus velezensis fermentation extract or fraction thereof. 7b. A skincare composition according to Embodiment 3, wherein the increase in keratinization occurs through 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 combination thereof, compared to the expression of the gene caused by a control composition lacking the Bacillus velezensis fermentation extract or fraction thereof. 8. A skincare composition according to Embodiment 1, further comprising additional compounds selected from the group consisting of fragrances, excipients, preservatives, pH adjusters, aqueous carriers, alcohol carriers, emollients, coagulants, hydrated agents, emulsifiers, solubilizers, surfactants, thickeners, salts, and any combination thereof. 9. Use of a skincare composition described in any of the prior embodiments in a cosmetic skincare product. 9b. Use of an effective amount of the skincare composition described in any of the prior embodiments in a cosmetic skincare product. 10. A skincare product comprising a skincare composition described in any of the prior embodiments and one or more dermatologically or skincare-acceptable components. 10b. A skincare product comprising a skincare composition for providing at least one skincare effect in a subject requiring it, wherein the composition comprises an effective amount of Bacillus velezensis ferment extract or a fraction thereof. 10c. A skincare product comprising one or more dermatologically or skincare-acceptable ingredients and a skincare composition for providing at least one skincare effect to a subject in need thereof, wherein the composition comprises an effective amount of Bacillus velezensis ferment extract or a fraction thereof, and the at least one skincare effect is selected from the group consisting of strengthening the skin barrier, improving the skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof. 10b. A skincare product comprising a skincare composition for providing at least one skincare effect in a subject requiring it, wherein the composition is manufactured by Westerdijk Fungal Biodiversity under the number CBS147469. Westerdijk Fungal Biodiversity has a B. velezensis strain, numbered CBS151099, with a 16S ribosomal RNA sequence exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the World Federation of Biodiversities Institute (WFDB). For the 16S ribosomal RNA sequence of B. velezensis E04_spo deposited at the Institute (WFDB), 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%, and 99.93% were obtained. A skincare product comprising a Bacillus velezensis ferment extract or fraction thereof, obtained from a Bacillus velezensis ferment selected from the group consisting of B. velezensis strains having 16S ribosomal RNA sequences exhibiting 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity, and any one combination thereof. 11. A skincare product according to Embodiment 10, comprising the skincare composition in an amount of at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% by weight relative to the total weight of the skincare product. 11b. A skincare product according to Embodiment 10, comprising at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% of the total volume of the skincare product, based on volume. 12. A skin care product according to Embodiment 10 or Embodiment 11, formulated for topical administration. 12b. A skin care product according to Embodiment 10 or 11, formulated for topical administration to the skin or scalp. 13. A skincare product according to any one of Embodiments 11 to 12, selected from the group consisting of lotions, serums, jellies, creams, gels, hydrogels, emulsions, solid cosmetics, masks, patches, shampoos, loose powders or compact powders, liquid suspensions or solutions, spray solutions and sticks. 14. A method for providing at least one skincare effect in a subject requiring it, comprising topically administering a skincare composition or skincare product described in any prior embodiment to the skin or scalp of the subject. 14b A method for providing at least one skin care effect to a subject in need thereof, comprising topically administering a skin care composition or a skin care product containing the composition to the skin of the subject, wherein the composition comprises an effective amount of B. velezensis ferment extract or a fraction thereof, and the at least one skin care effect is selected from the group consisting of strengthening the skin barrier, improving the skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof. 14c. A method for providing at least one skin care effect to a subject in need thereof, comprising topically administering a composition comprising an effective amount of B. velezensis ferment extract or a fraction thereof to the subject in need thereof, wherein the at least one skin care effect is selected from the group consisting of strengthening the skin barrier, improving the function of the skin barrier, moisturizing the skin, promoting skin repair, and any one combination thereof. 15. The method according to Embodiment 14, wherein the skincare product is selected from the group consisting of lotion, serum, jelly, cream, gel, hydrogel, emulsion, solid cosmetic, mask, patch, shampoo, loose powder or compact powder, liquid suspension or solution, spray solution and stick. 16. A method according to any one of Embodiments 14 to 15, wherein the B. velezensis fermentation extract is selected from the group consisting of cell-free supernatant obtained from B. velezensis ferment, whole broth fermentation extract obtained from B. velezensis ferment, and any combination thereof. 16b. A method according to any one of Embodiments 14 to 15, wherein the B. velezensis fermentation extract is selected from the group consisting of cell-free supernatant obtained from B. velezensis ferment, cell pellet extract from B. velezensis ferment, whole broth fermentation extract obtained from B. velezensis ferment, and any combination thereof. 16c. The method according to any one of Embodiments 14 to 15, wherein the Bacillus velezensis fermentation extract or fraction thereof is provided to Westerdijk Fungal Biodiversity under the number CBS147469. Westerdijk Fungal Biodiversity has a B. velezensis strain, numbered CBS151099, with a 16S ribosomal RNA sequence exhibiting at least 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.90%, 99.91%, 99.92%, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the World Federation of Biodiversities Institute (WFDB). For the 16S ribosomal RNA sequence of B. velezensis E04_spo deposited at the Institute (WFDB), 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%, and 99.92% were obtained. A method obtained from a B. velezensis fermentate selected from the group consisting of B. velezensis strains having 16S ribosomal RNA sequences exhibiting sequence similarity of %, 99.93%, 99.94%, 99.95%, 99.96%, 99.97%, 99.98%, 99.99%, and up to 100%, and any combination thereof. 16d. A method, skincare composition or skincare product for use according to any one of the prior embodiments, wherein the skincare product or skincare composition comprises at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% by weight of the skincare composition, selected from the group consisting of lotions, serums, jellies, creams, gels, emulsions, solid cosmetics, masks, patches and sticks. 17. A method for strengthening the skin barrier in a subject, comprising topically administering a skincare composition or skincare product described in any of the prior embodiments to the skin or scalp of the subject. 17b. A method for strengthening the skin barrier in a subject, comprising topically administering a skincare composition or skincare product containing an effective amount of B. velezensis ferment extract or a fraction thereof to the skin or scalp of the subject. 17c. A method for improving the skin barrier function of a subject, comprising topically administering a skincare composition or skincare product described in any of the prior embodiments to the skin or scalp of the subject. 17b. A method for improving the skin barrier function in a subject, comprising topically administering a skincare composition or skincare product containing an effective amount of B. velezensis ferment extract or a fraction thereof to the skin or scalp of the subject. 18. A method for moisturizing the skin of a subject, comprising topically administering a skincare composition or skincare product described in any of the prior embodiments to the skin or scalp of the subject. 18b. A method for moisturizing the skin of a subject, comprising topically administering a skincare composition containing an effective amount of B. velezensis ferment extract or a fraction thereof to the skin or scalp of the subject. 19. A method for increasing the activity of a fermented extract of Bacillus velezensis strain to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject in need thereof, (a) To provide a parent Bacillus velezensis strain capable of producing an effective amount of fermented extract to provide, in a subject requiring it, at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, (b) Modifying the parent Bacillus velezensis strain by knocking out at least one spore-forming gene from the genome of the Bacillus velezensis strain, thereby generating a variant strain that cannot form spores, (c) To produce a fermented extract of the Bacillus velezensis variant strain mentioned above. A method comprising the fermentation extract of the Bacillus velezensis variant strain having increased activity compared to the activity of the fermentation extract of the parent Bacillus velezensis strain, for providing at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject in need. 19b. A method for increasing the activity of a fermented extract of a Bacillus velezensis strain to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any combination thereof, to a subject in need thereof, comprising: (a) providing a parent Bacillus velezensis strain capable of producing an effective amount of fermented extract to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any combination thereof, to a subject in need thereof; (b) modifying the parent Bacillus velezensis strain by knocking out at least one spore-forming gene from the genome of the Bacillus velezensis strain, thereby generating a variant strain that cannot form spores; and (c) the Bacillus velezensis strain A method comprising producing a fermented extract of a Bacillus velezensis variant strain, wherein the fermented extract of the Bacillus velezensis variant strain has increased activity compared to the activity of the fermented extract of the parent Bacillus velezensis strain, for providing at least one skin care effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any combination thereof, to a subject in need thereof. 19c. In one embodiment, the present method is a method for increasing the activity of a fermented extract of a Bacillus velezensis strain to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject in need thereof, the method comprising (a) providing a parent Bacillus velezensis strain capable of producing an effective amount of fermented extract to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject in need thereof, and (b) knocking out at least one spore-forming gene from the genome of the Bacillus velezensis strain, thereby increasing the activity of the parent Bacillus velezensis strain (c) modifying the Bacillus velezensis strain to produce a variant strain that is incapable of forming spores, wherein at least one spore-forming gene is SpoIIE; and (c) producing a fermented extract of the Bacillus velezensis variant strain, wherein the fermented extract of the Bacillus velezensis variant strain has increased activity compared to the activity of the fermented extract of the parent Bacillus velezensis strain, for providing at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any one combination thereof, to a subject in need thereof. 20. A ferment of a Bacillus velezensis strain variant, a fermented extract of a Bacillus velezensis strain variant, or a fraction of the B. velezensis strain variant ferment, wherein the Bacillus velezensis strain The Bacillus velezensis strain variant is obtained by knocking out the spore-forming gene of the parent B. velezensis strain, and the variant ferment, the variant ferment extract, or the fraction of the variant ferment has increased activity compared to the activity of the ferment, ferment extract, or fraction thereof derived from the parent B. velezensis strain, and the activity is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof, and is a Bacillus velezensis strain variant ferment, Bacillus velezensis strain variant ferment extract, or the fraction of the variant ferment of the Bacillus velezensis strain variant. 20b. A ferment of a Bacillus velezensis strain variant, a fermented extract of a Bacillus velezensis strain variant, or a fraction of the B. velezensis strain variant ferment, wherein the Bacillus velezensis strain The Bacillus velezensis strain variant is obtained from the parent B. velezensis by knocking out the spore-forming gene of the parent B. velezensis strain, and the variant ferment, the variant ferment extract, or the fraction of the variant ferment has increased activity compared to the activity of the ferment, ferment extract, or fraction derived from the parent B. velezensis strain, and the activity is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, and the strengthening of the skin barrier, and / or the improvement of skin barrier function, and / or the moisturizing the skin occurs through an increase in tight junctions, an increase in desmosomes, an increase in cell-cell adhesion, an increase in keratinization, and any combination thereof, Bacillus velezensis strain variant ferment, Bacillus velezensis Fermentation extract of the B. velezensis strain variant or fraction of the variant ferment of the B. velezensis strain variant. [Examples]

[0168] In the following embodiments, unless otherwise specified, parts and percentages are by weight, and degrees are in Celsius. These embodiments illustrate embodiments of the present disclosure, but should be understood to be for illustrative purposes only. From the above discussion and these embodiments, those skilled in the art can make various modifications and alterations of the present disclosure to suit various uses and conditions. Such modifications are intended to be included within the scope of the appended claims.

[0169] In this specification, the following abbreviations correspond to the following units of measurement, methods, properties, or compounds: "sec" or "s" means seconds, "min" means minutes, "h" or "hr" means hours, "μL" means microliters, "mL" means milliliters, "L" means liters, "mM" means millimolar concentration, "M" means molar concentration, "mmol" means millimoles, "ppm" means parts per million, "wt" means weight, "wt%" means weight percent, "g" means grams, "mg" means milligrams, "μg" means micrograms, "ng" means nanograms, "conc." means concentration, and "Trt" means treatment.

[0170] Example 1 Bacillus velezensis strain A suitable Bacillus velezensis (B. velezensis) for use in this invention is the Bacillus velezensis deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under the number CBS147469. Examples of Bacillus velezensis include those having a 16S ribosomal RNA sequence that exhibits sequence similarity of 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% to the 16S ribosomal RNA sequence (SEQ ID NO: 1) of velezensis)E04.

[0171] A suitable Bacillus velezensis for use in this invention is Westerdijk Fungal Biodiversity under the number CBS151099. Bacillus velezensis (B. velezensis) E04_spo, deposited at the World Foundation for Food Research (WFDB) and in which the sporogenesis gene spoIIE is knocked out, has a 16S ribosomal RNA sequence that exhibits 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 the 16S ribosomal RNA sequence (SEQ ID NO: 1) of B. velezensis E04_spo, which has been deposited at the WFDB Institute and in which the sporogenesis gene spoIIE is knocked out. Velezensis is another example.

[0172] Example 2 Fermented Bacillus velezensis Bacillus velezensis strains were grown in TSB (trypsin soybean broth) medium supplemented with 1% glucose (TSG). TSG medium (1.0X intensity) consists of 17 g tryptone, 3 g soytone, 5 g sodium chloride, 10 g glucose, and 2.5 g dipotassium phosphate per liter. The TSG medium was adjusted to pH 7.3. B. velezensis E04 fermentation was carried out in a 125 mL flat-bottomed shaking flask containing 25 mL of medium. The flask was placed on a platform shaker with a constant speed of 200 rpm at 32°C. A fresh seed culture overnight was used as inoculum, and the starting OD was 0.125 at 600 nm. The culture was grown for 24 hours.

[0173] The Bacillus velezensis strain can also be grown in SUM (Soyptone Urea Micronutrient) medium. SUM medium (1.0x intensity) consists of 10 g of soyptone, 75 g of glucose, 3 mL of dipotassium phosphate (1M), 3.6 g of urea, and 10 mL of micronutrient stock per liter. The micronutrient stock contains 1.47 g of sodium citrate·2H2O, 1.47 g of CaCl2·2H2O, 0.4 g of FeSO4·7H2O, 0.1 g of MnSO4·H2O, 0.1 g of ZnSO4·H2O, 0.05 g of CuCl2·2H2O, 0.1 g of CoCl2·6H2O, and 0.1 g of Na2MoO4·2H2O per liter. SUM medium can be adjusted to pH 7.3. SUM medium may be used as the growth medium for B. velezensis as described herein, but B. velezensis strains may be grown in any other suitable medium that can support growth. In addition, incubation time may vary depending on the growth medium or growth method. It is understood that ferments may be collected at various incubation times and may still produce ferments containing an effective amount of Bacillus velezensis fermentation extract.

[0174] Example 3 Bacillus velezensis fermented extract An extract of the fermented Bacillus velezensis was obtained as follows.

[0175] Cell-free supernatant of Bacillus velezensis fermentation Cells were removed from the Bacillus velezensis ferment by pelletizing them through centrifugation at 4,000 g for 20 minutes. The resulting cell-free supernatant (fermentation extract) was sterilized by passing it through a 0.2 μM filter.

[0176] Cell pellet extract of Bacillus velezensis ferment Cells are pelletized (centrifuged at 4,000-8,000 × g), the supernatant is drained, and a portion of the cell pellet is left to obtain a cell pellet extract from the Bacillus velezensis ferment. The pellet is resuspended in acidic water (1 / 10 v / v) at pH 2.5-4.0, vortexed, pelletized again by centrifugation (4,000-8,000 × g), and the extract is passed through a 0.2 μM filter.

[0177] Starting with a cell pellet has the advantage of allowing the volume of the resuspension (extract volume) to be determined to produce an effective amount of active extract, and / or further concentration to produce an effective amount of active extract. Furthermore, by resuspending the pellet in water or non-fermented broth liquid, non-effective components of the fermented broth that may interfere with the active substances in the cell pellet extract are reduced or removed.

[0178] Combination of cell-free supernatant and cell pellet extract from Bacillus velezensis ferment Bacillus velezensis fermentation extract can also be produced by combining the cell-free supernatant fraction described above with the cell pellet extract described above.

[0179] Whole Brosbacillus velezensis fermented extract To obtain a whole broth ferment extract, the pH of the ferment (whole fermented broth - Example 2) is first adjusted to pH 2.5-4.0, then the insoluble cytomaterial is pelleted, and the supernatant is filtered through a 0.2 μM filter to obtain a pH-adjusted cell-free supernatant, thereby preparing the Bacillus velezensis ferment extract. Alternatively, the Bacillus velezensis whole broth ferment extract is prepared by first adjusting the pH of the ferment (whole fermented broth) to an alkaline pH, then the insoluble cytomaterial is pelleted, and the supernatant is optionally filtered through a 0.2 μM filter to obtain a cell-free supernatant (also called "whole broth ferment extract").

[0180] Example 4 Improving skin barrier function using Bacillus velezensis fermented extract. The ability of Bacillus velezensis fermentation extract to improve and strengthen skin barrier function was evaluated. B. velezensis strain E04 (deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under the number CBS147469) was grown in TSB (trypsin soybean broth) medium (TSG) supplemented with 1% glucose. TSG medium (1.0X intensity) consists of 17 g tryptone, 3 g soytone, 5 g sodium chloride, 10 g glucose, and 2.5 g dipotassium phosphate per liter. The TSG medium was adjusted to pH 7.3. B. velezensis E04 fermentation was carried out in a 125 mL flat-bottomed shaking flask containing 25 mL of medium. The flask was placed in a platform shaker with a constant speed of 200 rpm at 32°C. Fresh seed cultures prepared overnight were used as inoculum, with a starting OD of 0.125 at 600 nm. After 24 hours of growth, the fermentation extract (cell-free supernatant) was obtained by centrifugation and filtration sterilization of the ferment.

[0181] Normal human epidermal keratinocytes (NHEKs) isolated from one adult donor were thawed in Keratinocyte Growth Medium 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), epinephrine (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). Subsequently, the NHEKs were cultured in a T75 flask containing KGM2 and reached 75-80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, the NHEKs were removed from the T75 flask using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK cells were resuspended in KGM2, counted using a hemocytometer, and seeded onto Transwell inserts (6.5 mm insert, 0.4 μm polyester membrane, tissue culture treatment, polystyrene 24-well plate). NHEK cells were incubated in a 37°C incubator supplied with 5% CO2 and 95% humidity until they reached 100% confluence on the Transwell membrane. At that point, the medium was aspirated, and NHEK cells were subsequently grown in KGM2 supplemented with 1.5 mM calcium chloride instead of 0.06 mM for the remainder of the experiment. B. velezensis E04 fermentation extract was added to the upper wells to a final fermentation extract concentration of 1%. Transcutaneous electrical resistance (TEER) was measured daily using an epithelial bolt / ohmmeter (EVOM) after the addition of the fermentation extract. The medium and fermentation extract were replaced every two days. The results are shown in Table 1.

[0182] [Table 1]

[0183] The results in Table 1 show that the addition of 1% B. velezensis E04 fermented extract resulted in a higher percentage increase in TEER values ​​compared to the untreated control, which is an indicator of improved skin barrier function and strengthening of the skin barrier.

[0184] Example 5 Improvement of skin barrier function using a fraction of B. velezensis fermented extract The ability of specific molecular weight fractions of B. velezensis fermented extract to improve and strengthen skin barrier function was evaluated. Fermented extract (cell-free supernatant) from B. velezensis E04 was prepared as described in Example 4. After centrifugation and filtration sterilization of the ferment to obtain the fermented extract, the fermented extract was fractionated by molecular weight by passing it through a series of molecular weight cutoff filters in the following order: 100 kilodaltons (kDa), 50 kDa, 30 kDa, and 10 kDa. The fermented extract that did not pass through the filters was saved and called the retention solution (R), and the fermented extract that passed through the filters was saved and called the flow-through (FT). Each B. velezensis E04 molecular weight retention solution and flow-through was tested for its ability to improve skin barrier function using NHEK, as performed in Example 1. The results are shown in Table 2.

[0185] [Table 2]

[0186] The results in Table 2 show that when a 1% B. velezensis E04 fermented extract, filtered through molecular weight cutoff filters of 100 kDa, 50 kDa, and 30 kDa, was added, the TEER value increased by a higher percentage compared to the untreated control. This is an indicator of improved and strengthened skin barrier function.

[0187] Example 6 Strengthening of the skin barrier through tight junctions using fermented B. velezensis extract. The mechanism by which B. velezensis fermentation extract affects the keratinocyte barrier was evaluated. Normal human epidermal keratinocytes (NHEKs) isolated from one adult donor were thawed in Keratinocyte Growth Medium 2 (KGM2) supplemented with bovine pituitary gland extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), epinephrine (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). Subsequently, the NHEKs were cultured in a T75 flask containing KGM2 and reached 75 to 80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, NHEK was removed from the T75 flask using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK was resuspended in KGM2, counted using a hemocytometer, and seeded into a 96-well plate. A fermentation extract (cell-free supernatant) from B. velezensis E04 was prepared 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 the B. velezensis E04 fermentation extract for 18 hours. After stimulation with the B. velezensis E04 fermentation extract, RNA was isolated from NHEK using the RNAqueous Micro Kit. RNA was further processed using the Illumina Stranded mRNA prep kit to extract mRNA from all other RNA types. Individual mRNA libraries were normalized to 2nM, pooled, and 4% phiX was added to the pooled mRNA libraries. The 1nM pooled libraries were loaded onto either a P1, P2, or P3 Illumina chip, and then sequenced using either an Illumina NextSeq 1000 or Illumina NextSeq 2000 instrument.Tables 3A-3B show 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) in NHEK of the control and B. velezensis fermentation extract-treated samples.

[0188] [Table 3A]

[0189] [Table 3B]

[0190] The results shown in Tables 3A-3B demonstrate that the B. velezensis E04 fermentation extract increases the expression of the following genes in NHEK: 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). This indicates that the B. velezensis E04 fermentation extract strengthens the keratinocyte barrier through an increase in tight junctions, and is therefore suitable for use in strengthening the skin barrier, improving skin barrier function, and moisturizing the skin.

[0191] Example 7 Strengthening of the skin barrier through desmosome-mediated fermentation extract of B. velezensis The mechanism by which B. velezensis fermentation extract affects the keratinocyte barrier was analyzed. Normal human epidermal keratinocytes (NHEK) isolated from one adult donor were thawed in Keratinocyte Growth Medium 2 (KGM2) supplemented with bovine pituitary gland extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), epinephrine (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). Subsequently, the NHEK were cultured in a T75 flask containing KGM2 and reached 75-80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, NHEK was removed from the T75 flask using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK was resuspended in KGM2, counted using a hemocytometer, and seeded into a 96-well plate. A fermentation extract (cell-free supernatant) from B. velezensis E04 was prepared 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 the B. velezensis E04 fermentation extract for 18 hours. After stimulation with B. velezensis E04, RNA was isolated from NHEK using the RNAqueous Micro Kit. RNA was further processed using the Illumina Stranded mRNA prep kit to extract mRNA from all other RNA types. Individual mRNA libraries were normalized to 2nM, pooled, and 4% phiX was added to the pooled mRNA libraries. The 1nM pooled libraries were loaded onto either a P1, P2, or P3 Illumina chip, and then sequenced using either an 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 (desmocolin 2, gene ID 1824), EVPL (emboplakin, gene ID 2125), DSG3 (desmogelin 3, gene ID 1830), DSP (desmoplakin, gene ID 1832), and DSC3 (desmocolin 3, gene ID 1825) in control NHEK and B. velezensis fermentation extract-treated samples.

[0192] [Table 4]

[0193] The results shown in Table 4 indicate that the fermented extract of B. velezensis E04 increases the expression of 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. This demonstrates that B. velezensis E04 strengthens the keratinocyte barrier through desmosome increase, and is therefore suitable for use in strengthening the skin barrier, improving skin barrier function, and moisturizing the skin.

[0194] Example 8 Strengthening of the skin barrier through cell-cell adhesion using B. velezensis fermented extract The mechanism by which B. velezensis fermentation extract affects the keratinocyte barrier was analyzed. Normal human epidermal keratinocytes (NHEK) isolated from one adult donor were thawed in Keratinocyte Growth Medium 2 (KGM2) supplemented with bovine pituitary gland extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), epinephrine (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). Subsequently, the NHEK were cultured in a T75 flask containing KGM2 and reached 75-80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, NHEK was removed from the T75 flask using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK was resuspended in KGM2, counted using a hemocytometer, and seeded into a 96-well plate. Fermentation extract (cell-free supernatant) from B. velezensis E04 was prepared 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 the B. velezensis E04 fermentation extract for 18 hours. After stimulation with B. velezensis E04, RNA was isolated from NHEK using the RNAqueous Micro Kit. RNA was further processed using the Illumina Stranded mRNA prep kit to extract mRNA from all other RNA types. Individual mRNA libraries were normalized to 2nM, pooled, and 4% phiX was added to the pooled mRNA libraries. The 1nM pooled libraries were loaded onto either a P1, P2, or P3 Illumina chip, and then sequenced using either an Illumina NextSeq 1000 or Illumina NextSeq 2000 instrument.Tables 5A-5B show the 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 the control and B. velezensis fermentation extract-treated samples.

[0195] [Table 5A]

[0196] [Table 5B]

[0197] The results given in Tables 5A-5B indicate that B. velezensis E04 is NHEK-positive and has 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), and NECTI The study demonstrated that B. velezensis E04 fermented extract increases the expression of N1 (gene ID 5818), PCDHGA9 (gene ID 56107), PCDHB14 (gene ID 56122), PCDHGA11 (gene ID 56105), and PCDHGA12 (gene ID 26025), indicating that B. velezensis E04 fermented extract enhances the keratinocyte barrier through increased cell-cell adhesion, and is therefore suitable for use in strengthening the skin barrier, improving skin barrier function, and moisturizing the skin.

[0198] Example 9 Improvement of the skin barrier (cornified envelope) through keratinization by fermented extract of B. velezensis. The mechanism by which B. velezensis fermentation extract affects the keratinocyte barrier was analyzed. Normal human epidermal keratinocytes (NHEK) isolated from one adult donor were thawed in Keratinocyte Growth Medium 2 (KGM2) supplemented with bovine pituitary gland extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), epinephrine (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). Subsequently, the NHEK were cultured in a T75 flask containing KGM2 and reached 75-80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, NHEK was removed from the T75 flask using trypsin and centrifuged at room temperature (RT) and 1500 RPM for 5 minutes. NHEK was resuspended in KGM2, counted using a hemocytometer, and seeded into a 96-well plate. Fermentation extract (cell-free supernatant) from B. velezensis E04 was prepared 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 the B. velezensis E04 fermentation extract for 18 hours. After stimulation with B. velezensis E04, RNA was isolated from NHEK using the RNAqueous Micro Kit. RNA was further processed using the Illumina stranded mRNA prep kit to extract mRNA from all other RNA types. Individual mRNA libraries were normalized to 2nM, pooled, and 4% phiX was added to the pooled mRNA libraries. The 1nM pooled libraries were loaded onto either a P1, P2, or P3 Illumina chip, and then sequenced using either an Illumina NextSeq 1000 or Illumina NextSeq 2000 instrument.Tables 6A-6B show 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 the control and B. velezensis fermentation extract-treated samples.

[0199] [Table 6A]

[0200] [Table 6B]

[0201] The results shown in Tables 6A-6B demonstrate that B. velezensis E04 increases 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. This indicates that B. velezensis E04 strengthens the keratinocyte barrier through increased keratinization, and is therefore suitable for use in strengthening the skin barrier, improving skin barrier function, and moisturizing the skin.

[0202] Example 10 Knocking out the B. velezensis sporogenesis gene spoIIE further enhances the ability of B. velezensis fermented extract to improve skin barrier function. The B. velezensis strain contains genes associated with spore formation, such as SpollE (Sequence ID 2, Barak et al. 1996, Molecular Microbiology 19(5), 1047-1060). In one embodiment, elimination of the spollE gene is desired, particularly in B. velezensis-producing strains.

[0203] We evaluated the ability of a fermented extract of B. velezensis E04 spore-forming spoIIE knockout (referred to as B. velezensis E04_spo, CBS151099) to improve skin barrier function.

[0204] A CRISPR-Cas9 plasmid (referred to as pMY2-3) was constructed to enable deletion of the spoIIE locus from B. velezensis E04 (CBS147469), thereby inhibiting its ability to produce spores. The pMY2-3 plasmid contained an edit template insert (SEQ ID NO: 3) containing a Streptococcus pyogenes Cas9 endonuclease expression cassette and a B. velezensis E04 homologous region, sgRNA (SEQ ID NO: 4), and a kanamycin-resistant cassette. The pMY2-3 plasmid was introduced into Endura® electrocompetent cells (Biosearch Technologies) by electroporation. The assembled construct was validated by Sanger sequencing. pMY2-3 was amplified using rolling circle amplification (RCA) with a GenomiPhi HY Ready-to-Go DNA amplification kit (Cytiva).

[0205] B. velezensis E04 (CBS147469) was modified to contain the competence plasmid pBL.com K and spoIIE gene deletion described in International Publication No. 2019 / 40423. Briefly, B. velezensis E04 was transformed with pBL.com K using natural competence and spectinomycin as a resistance marker. The pBL.com K-containing strain was then made competent as described in International Publication No. 2021 / 146411 and subsequently transformed with plasmid pMY2-MY3. 200 μL of competent cells were mixed with 50 μL of pMY2-3 RCA product and 1 mL of Luria-Bertani (LB) broth, and then incubated at 30°C and 250 RPM for 1.5 hours. The mixture was plated onto LB agar plates containing 20 ppm kanamycin, and cells transformed with Cas9 plasmid pMY2-3 were selected. Isolates were screened for spoIIE deletion using Q5® PCR with primers BVspo-checkdel-F (SEQ ID NO: 5) and BVspo-checkdel-R (SEQ ID NO: 6). Colonies with spoIIE deletion (ΔspoIIE) produced a PCR product of 2253 bp, while wild-type strains containing the intact spoIIE gene produced a PCR product of 4743 bp. The deletion was confirmed via Sanger sequencing, and the confirmed mutant isolates were stored as glycerol preserves at -80°C.

[0206] The isolated strains were cultured in antibiotic-free trypsin-soybean broth (TSB) for 3 days and subcultured into fresh TBS once every 24 hours to restore pBL.comK and pMY2-3 in the isolated strains, which were then plated onto antibiotic-free trypsin-soybean agar (TSA). Colonies were selected from the TSA and patched onto antibiotic-free plain LB agar plates, LB agar plates containing 20 ppm kanamycin, and LB agar plates containing 100 ppm spectinomycin. The restored isolated strains grew on plain LB agar but not on LB containing kanamycin or spectinomycin.

[0207] After confirming the genotype of the spoIIE deletion, phenotypic analysis using heat treatment was performed to confirm the loss of spore-forming ability. B. velezensis E04 was grown in Difco spore-forming medium (DSM) to promote spore formation (see Nicholson, W.L. and Setlow, P., 1990, Sporulation, germmination and outgrowth. In Molecular Biological Methods for Bacillus, edited by C.Harwood and S.M.C. Cutting, John Wiley & Sons, Chichester, UK, pp. 391-450). Wild-type and ΔspoIIE clones were freshly plated onto TSA, and single colonies were inoculated into DSM and incubated at 32°C and 200 RPM for 3 days. Two 100 μL aliquots were taken from each culture and transferred to 1.5 mL microcentrifuge tubes. The test aliquots were heated at 90°C for 15 minutes, while the control aliquots were left at room temperature (RT). Next, the entire 100 μL aliquot was plated onto TSA and incubated overnight at 32°C. Spore-positive isolates, such as wild-type B. velezensis E04, showed growth under both room temperature (RT) and 90°C treatment. Spore-negative isolates showed growth under RT but not at 90°C. Spore-negative clones (also called B. velezensis E04_spo strain) were stored as glycerol stocks at -80°C.

[0208] B. velezensis E04 (CBS147469, also known as B. velezensis E04 wild type (WT)) and B. velezensis E04_spo strains were grown in TSB (Tripty Soy Broth) medium supplemented with 1% glucose (TSG). TSG medium (1.0X intensity) consists of 17 g tryptone, 3 g soytone, 5 g sodium chloride, 10 g glucose, and 2.5 g dipotassium phosphate per liter. The TSG medium was adjusted to pH 7.3. B. velezensis E04 was grown in a 125 mL flat-bottomed shaking flask containing 25 mL of TSG. WT and KO ferments were prepared. Flasks were placed in a platform shaker with a constant speed of 200 rpm at 32°C. A fresh seed culture prepared overnight was used as inoculum, with a starting OD of 0.125 at 600 nm. After 24 hours of growth, the fermentation extract (cell-free supernatant) was obtained by centrifugation and filtration sterilization of the ferment.

[0209] Normal human epidermal keratinocytes (NHEKs) isolated from one adult donor were thawed in Keratinocyte Growth Medium 2 (KGM2) supplemented with bovine pituitary extract (0.0004 ml / ml), epidermal growth factor (0.125 ng / ml), insulin (5 μg / mL), hydrocortisone (0.33 μg / mL), epinephrine (0.39 μg / mL), transferrin (10 μg / mL), and calcium chloride (0.06 mM). Subsequently, the NHEKs were cultured in a T75 flask containing KGM2 and reached 75-80% confluence in a 37°C incubator supplied with 5% CO2 and 95% humidity. After reaching the desired confluence, the NHEKs were removed from the T75 flask using trypsin and centrifuged at 1500 RPM for 5 minutes at room temperature (RT). NHEK cells were resuspended in KGM2, counted using a hemocytometer, and seeded in Transwell inserts (6.5 mm inserts, 0.4 μm polyester membrane, tissue culture treated, polystyrene 24-well plates). The NHEK cells were incubated in a 37°C incubator supplied with 5% CO2 and 95% humidity until they reached 100% confluence on the Transwell membrane. At that point, the medium was aspirated with NHEK, and the cells were subsequently grown in KGM2 supplemented with 1.5 mM calcium chloride instead of 0.06 mM for the remainder of the experiment. B. velezensis fermentation extract was added to the upper wells to a final fermentation extract concentration of 0.5%. Transcutaneous electrical resistance (TEER) was measured daily using an epithelial bolt / ohmmeter (EVOM) after the addition of the fermentation extract. The medium and fermentation were refreshed every two days. The results are shown in Table 7.

[0210] [Table 7]

[0211] The results in Table 7 show that the addition of 0.5% of B. velezensis E04_spo fermentation extract (cell-free supernatant) resulted in a higher percentage increase in the TEER value compared to the addition of 0.5% of B. velezensis E04, indicating a further improvement in barrier function compared to the untreated control sample.

[0212] Example 11 Fermented extract of B. velezensis for promoting skin repair The ability of B. velezensis fermentation extract to promote skin repair was evaluated. B. velezensis strain E04 was grown in TSB (trypsin soybean broth) (TSG) medium supplemented with 1% glucose. TSG medium (1.0X intensity) consists of 17 g tryptone, 3 g soytone, 5 g sodium chloride, 10 g glucose, and 2.5 g dipotassium phosphate per liter. The TSG medium was adjusted to pH 7.3. B. velezensis E04 fermentation was carried out in a 125 mL flat-bottomed shaking flask containing 25 mL of medium. The flask was placed in a platform shaker with a constant speed of 200 rpm at 32°C. A fresh seed culture prepared overnight was used as inoculum, with a starting OD of 0.125 at 600 nm. After 24 hours of growth, the fermentation extract (cell-free supernatant) was obtained by centrifugation and filtration sterilization of the fermentation.

[0213] HaCaT cells were thawed in DMEM (complete medium) supplemented with 10% FBS and cultured in T75 flasks in a 37°C incubator supplied with 5% CO2 until 90% confluence was reached. After confluence was reached, HaCaT cells were removed from the flask using trypsin, centrifuged at room temperature for 5 minutes, resuspended in complete medium, and counted using a hemocytometer. Subsequently, HaCaT cells were seeded into 24-well plates and incubated in a 37°C incubator supplied with 5% CO2 for 48 hours until the cells could reach 100% confluence. Scratches were then made in the confluent HaCaT cell layer, the cells were washed with 1×PBS, and then returned to the wells either supplemented with 1% B. velezensis E04 fermentation extract or left unsupplemented with complete medium. Cells were incubated overnight in a 37°C incubator supplied with 5% CO2, and then imaged to assess scratch repair / closure. The results are shown in Table 8.

[0214] [Table 8]

[0215] The results in Table 8 show that treatment of keratinocytes with B. velezensis E04 fermented extract increased closure in the scratch test assay (smaller mean scratch) (100% closure observed in Table 8), indicating that B. velezensis E04 fermented extract promotes skin repair.

[0216] Example 12 Fermented extract fraction of B. velezensis for promoting skin repair The ability of specific molecular weight fractions of B. velezensis fermentation extract (cell-free supernatant) to promote skin repair was evaluated. Fermentates were produced from B. velezensis E04 using the method described in Example 11. After centrifugation and filtration sterilization of the fermentates, cell-free supernatant fermentation extracts were produced. The fermentation extracts were then fractionated by molecular weight by passing them through a series of molecular weight cutoff filters in the following order: 100 kilodaltons (kDa), 50 kDa, 30 kDa, and 10 kDa. The fermentation extracts that did not pass through the filters were saved and named retained (R), and the fermentation extracts that passed through the filters were saved and named flow-through (FT). As performed in Example 11, each B. velezensis E04 molecular weight retained solution and flow-through were tested for their ability to improve skin repair using HaCa T cells. The results are shown in Table 9.

[0217] [Table 9]

[0218] The results in Table 9 reveal that treating keratinocytes with a fermented extract fraction of B. velezensis E04 containing components greater than 100 kDa increased closure in the scratch test assay (resulting in smaller average scratches), indicating that components greater than 100 kDa produced by B. velezensis E04 promote skin repair.

[0219] Example 13 The bioactive flagellin in the fermented extract of B. velezensis is involved in skin repair. To identify the active ingredients of B. velezensis, which improves skin repair, the active molecular weight fraction (100 kDa retained solution) was separated on a 4-12% polyacrylamide gel, and protein bands were visualized by Coomassi staining. To extract the proteins, each separate band was excised from the gel and heated overnight in 1×TBS. The supernatant was collected and then processed for mass spectrometry. Peptide fragments were collected, and all proteins in the corresponding bands were identified using BLASTx. Flagellin was identified as the most abundant protein in the skin repair active molecular weight fraction (100 kDa retained solution) and was therefore tested by scratch assay.

[0220] Flagellin from B. velezensis was purified by collecting 1.5 ml of the overnight culture by centrifugation at 12 K rpm for 2 minutes. The cells were washed three 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 vigorously agitated for 2 minutes. After centrifugation at 12 K rpm for 2 minutes in the microcentrifuge tube, the supernatant containing flagellin was collected. To confirm the isolation of flagellin, the supernatant was separated on a 4-12% polyacrylamide gel and protein bands were identified using Coomasie. Only one band corresponding to 37 kDa, the size of the flagellin monomer, was visualized. In its natural form, flagellin is constructed as an oligomer with a molecular weight exceeding 100 kDa.

[0221] As performed in Example 11, purified B. velezensis E04 flagellin was tested for its ability to improve skin repair using HaCaT cells. The results are shown in Table 10.

[0222] [Table 10]

[0223] The results in Table 10 show that treatment of keratinocytes with purified flagellin from B. velezensis E04 increased closure in the scratch test assay (the average number of scratches measured was smaller), thereby indicating that flagellin produced by B. velezensis E04 and present in the fermented extract of B. velezensis E04 promotes skin repair.

Claims

1. A skincare composition for providing at least one skincare effect to a subject in need thereof, comprising an effective amount of Bacillus velezensis ferment extract or fraction thereof, wherein the at least one skincare effect is selected from the group consisting of strengthening the skin barrier, improving the skin barrier function, moisturizing the skin, promoting skin repair, and any one combination thereof.

2. The skincare composition according to claim 1, wherein the Bacillus velezensis fermentation extract is selected from the group consisting of cell-free supernatant obtained from Bacillus velezensis ferment, whole broth fermentation extract obtained from Bacillus velezensis ferment, and any one combination thereof.

3. The skincare composition according to claim 1, wherein the strengthening of the skin barrier and / or the improvement of the skin barrier function and / or the moisturizing of the skin occurs through an increase in tight junctions, an increase in desmosomes, an increase in cell-cell adhesion, an increase in keratinization, and any combination thereof.

4. The skincare composition according to claim 3, wherein the increase in tight junctions occurs through 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 combination thereof, compared to the expression of the gene caused by a control composition lacking the effective amount of the Bacillus velezensis fermentation extract or fraction thereof.

5. The skincare composition according to claim 3, wherein the increase in desmosomes occurs through 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 combination thereof, compared to the expression of the gene caused by a control composition lacking the Bacillus velezensis fermentation extract or fraction thereof.

6. The increase in cell-cell adhesion is due to genes 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), PCDH15 (gene ID 56121), PCDHGB6 (gene ID 56100), CADM4 (gene ID 199731), NECTIN1 (gene ID 5818), PCDHGA9 (gene ID 56107), PCDH14 (gene ID 56122), PCDHGA11 (gene ID 56105), and PCDHGA12 (gene ID 26025), and any combination thereof, in Bacillus berezensis. The skincare composition according to claim 3, which is caused by increased expression of the gene compared to the gene expression caused by a control composition lacking a fermented extract of velezensis or a fraction thereof.

7. The skincare composition according to claim 3, wherein the increase in keratinization occurs through 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 combination thereof, compared to the expression of the gene caused by a control composition lacking the effective amount of the Bacillus velezensis fermentation extract or fraction thereof.

8. The skincare composition according to claim 1, further comprising additional compounds selected from the group consisting of fragrances, excipients, preservatives, pH adjusters, aqueous carriers, alcohol carriers, emollients, coagulants, hydrates, emulsifiers, solubilizers, surfactants, thickeners, salts, and any combination thereof.

9. Use of the skincare composition according to any one of claims 1 to 8 in a beauty skincare product.

10. A skincare product comprising the skincare composition according to any one of claims 1 to 8 and one or more dermatologically or skincare-acceptable ingredients.

11. The skincare product according to claim 10, comprising at least 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and up to 10% by weight of the skincare composition relative to the total weight of the skincare product.

12. A skin care product according to claim 10 or 11, formulated for topical administration.

13. A skincare product according to claim 11 or 12, selected from the group consisting of lotions, serums, jellies, creams, gels, hydrogels, emulsions, solid cosmetics, masks, patches, shampoos, loose powders or compact powders, liquid suspensions or solutions, spray solutions, and sticks.

14. A method for providing at least one skincare effect to a subject in need thereof, comprising topically administering a skincare composition or skincare product according to any one of claims 1 to 8 or 10 to 13 to the skin or scalp of the subject.

15. The method according to claim 14, wherein the skincare product is selected from the group consisting of lotion, serum, jelly, cream, gel, hydrogel, emulsion, solid cosmetic, mask, patch, shampoo, loose powder or compact powder, liquid suspension or solution, spray solution and stick.

16. The method according to claim 14 or 15, wherein the B. velezensis fermentation extract is selected from the group consisting of cell-free supernatant obtained from B. velezensis ferment, whole broth fermentation extract obtained from B. velezensis ferment, and any one combination thereof.

17. A method for strengthening the skin barrier in a subject, comprising topically administering a skincare composition or skincare product according to any one of claims 1 to 8 or 10 to 13 to the skin or scalp of the subject.

18. A method for moisturizing the skin of a subject, comprising topically administering a skincare composition or skincare product according to any one of claims 1 to 8 or 10 to 13 to the skin or scalp of the subject.

19. A method for increasing the activity of a fermented extract of Bacillus velezensis strain to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject in need thereof, (a) To provide a parent Bacillus velezensis strain capable of producing an effective amount of fermented extract to provide at least one skincare effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject requiring it, (b) Modifying the parent Bacillus velezensis strain by knocking out at least one spore-forming gene from the genome of the Bacillus velezensis strain, thereby generating a variant strain that cannot form spores, (c) Producing a fermentation extract of the Bacillus velezensis variant strain mentioned above. A method comprising the fermentation extract of the Bacillus velezensis variant strain having increased activity compared to the activity of the fermentation extract of the parent Bacillus velezensis strain, for providing at least one skin care effect selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any combination thereof, to a subject requiring such effect.

20. A ferment of a Bacillus velezensis strain variant, a fermented extract of a Bacillus velezensis strain variant, or a fraction of the variant ferment of a B. velezensis strain variant, wherein the Bacillus velezensis strain variant is obtained by knocking out the spore-forming gene of the parent B. velezensis strain, and the variant ferment, the variant fermented extract, or the fraction of the variant ferment is obtained from the parent B. A ferment product, ferment extract, or fraction of a Bacillus velezensis strain variant having increased activity compared to the activity of a ferment product, ferment extract, or fraction thereof derived from the Bacillus velezensis strain, wherein the activity is selected from the group consisting of strengthening the skin barrier, improving skin barrier function, moisturizing the skin, and any one combination thereof, and is a ferment product of a Bacillus velezensis strain variant, a ferment extract of a Bacillus velezensis strain variant, or a fraction of the variant ferment product of the Bacillus velezensis strain variant.

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