Methods and compositions for microbial treatment of skin disorders

Abstract

The present disclosure is directed to skin care compositions, skin care formulations, and methods for providing treatment for scalp disorders. More specifically, the present disclosure is directed to methods and compositions comprising Bacillus velezensis ferment and / or metabolites thereof for treating scalp disorders, including dandruff.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 119701, filed December 1, 2020, and U.S. Provisional Patent Application No. 63 / 154903, filed March 1, 2021, each of which is incorporated by reference in its entirety herein.

[0002] The present disclosure is directed to skin care compositions, skin care formulations and methods for providing treatment for scalp disorders. More specifically, the present disclosure is directed to methods and compositions comprising Bacillus velezensis ferment and / or metabolites thereof for treating scalp disorders, including dandruff.

[0003] Reference to Electronically Submitted Sequence Listing The contents of Sequence Listing 20211118_NB41905PCT_ST25 (size: 12,102 bytes; creation date: November 18, 2021), submitted electronically with this application as an ASCII text file, are incorporated herein by reference in their entirety. [Background technology]

[0004] The skin not only protects the organism from drying out, but also acts as a barrier protecting the organism from the intrusion of foreign, often harmful, substances.

[0005] The skin is also home to a diverse community of microorganisms, the majority of which are commensal (non-pathogenic resident) or transient (temporarily resident) organisms. When interacting with pathogens, only the microbes benefit, while the host is ultimately harmed. Many skin pathogens that live on the skin can typically be seen as commensals, but can change from commensal to pathogenic forms triggered by microbiota disturbances (or microbial imbalances), host genetic mutations and immune status (Findley, K. and Grice, EA, The Skin Microbiome: A Focus on Pathogens and Their Association with Skin Disease. PLoS Pathog. 2014, 10).

[0006] The epidermis constitutes the outermost region of the skin tissue and forms as such the actual protective envelope against the environment. The outer layer of the epidermis (Stratum corneum or Horny layer) is the part in contact with the environment and its specific structure protects the skin and stabilizes its flexibility by binding a defined amount of water (PMElias, Drug Dev. Res. 13, 1988, 97-105).

[0007] Spatially, the skin microbiota may extend to the subepidermal compartments (Nakatsuji, T et al., The Microbiome Extends to Subepidermal Compartments of Normal Skin. Nat. Commun. 2013, 4). Areas with a high density of sebaceous glands, such as the face, chest and back, activate the proliferation of fat-loving microorganisms, such as Propionibacterium and Malassezia.

[0008] Skin is a unique environment in which microorganisms often exist as biofilms (Brandwein, et al., 2016. NPJ Biofilms Microbiomes 2:3). Biofilms can form on the epithelial surface of the skin or inside hair follicles. Biofilms consist of cells as well as extracellular components such as exopolysaccharides, proteins and DNA. This complex structure can provide a physical and chemical barrier to certain compounds. However, more importantly, the physiology of microorganisms in a biofilm state is significantly different from that of planktonic microorganisms. This is especially true with regard to their ability to combat environmental stresses and resist various antimicrobial treatments (Koo, et al., Nature Reviews Microbiology 15:740-755, 2017).

[0009] Malassezia species, yeasts isolated from both healthy and unhealthy skin, have been shown to form biofilms in vitro (Angiolella, et al. 2020, Med Mycol. 0:1-7). Such isolates of Malassezia globosa (M. globosa) can not only form biofilms but also exhibit very high adherence and / or hydrophobicity. Biofilm forms of Malassezia species have been shown to significantly reduce their susceptibility to antifungal agents (Figueredo, et al., 2016, Medical Mycology 8:863-867, 2013; Bumroogthai, et al., Medical Mycology 54:544-549). Biofilm adherence and hydrophobicity have been suggested to be virulence factors for Malassezia (Allen, et al., 2018, J. of Clinical & Experimental Dermatology Research 6:311, 2015; Angiolella, et al., Medical Mycology 56:110-116). Strategies to eliminate Malassezia biofilms may therefore be beneficial in the treatment of various skin conditions caused by this group of organisms.

[0010] Malassezia is the major fungus of the skin microbiota, found on almost everyone's scalp and responsible for the most common skin disorders, including but not limited to seborrheic dermatitis, dandruff and tinea versicolor. Dandruff is the term commonly used for seborrhea of ​​the scalp. It is mainly Malassezia species such as Malassezia restricta (M.restricta) and Malassezia globosa (M.globosa) that are involved, and the prevalence is very high, almost 50% of the population (Schommer,NN;Gallo,RL,Structure and Function of the Human Skin Microbiome.Trends Microbiol.2013,21,660-668). The application of antifungal agents, rather than antibacterial agents, in treatment can improve the disease. The underlying pathogenic mechanisms are not fully understood. Impaired skin barrier function promotes the disease process (Harding, CR et al., Dandruff: a condition characterized by decreased levels of intercellular lipids in scalp stratum corneum and impaired barrier function. Arch. Dermatol. Res. 2002, 294, 221-230).

[0011] Malassezia species lack fatty acid synthases and must rely on sebum lipids for a carbon source. They also lack delta 2,3-enoyl-CoA isomerase to effectively utilize unsaturated FAs (e.g., oleic acid). Malassezia species feed on sebum lipids (by secreting one or more lipases that break down triglycerides into irritating fatty acids), and as the lipids in sebum are broken down, fatty acids (such as oleic acid) are released as by-products. Many people are sensitive to free fatty acids because they can induce hyperproliferation and scaling or the release of arachidonic acid, which is also involved in inflammation, to which the scalp responds with irritation. In response to this irritation, the scalp begins to swell, become red, and itch, and skin cells slough off the body more quickly than normal as the body tries to remove the source of irritation. This sloughing causes visible flakes to appear on the scalp. This is dandruff.

[0012] In addition to the yeast Malassesia species, Staphylococcus species such as S. aureus or S. epidermidis are associated with dandruff scalp, while Propionibacterial species are associated with healthy scalp (Saxena, et al., 2018, Front Cell Infect Microbiol, 8:346; Xu, et al. 2016, Sci Rep., 6:24877). Inhibiting certain populations of Staphylococcus may be beneficial in treating dandruff. Certain Staphylococcus species, including S. aureus, can form biofilms (Crit Rev Microbiol, 43:602-620, 2017). Summary of the Invention [Problem to be solved by the invention]

[0013] There remains a need to find methods and skin care compositions for providing treatment for scalp disorders, including, but not limited to, methods and compositions for treating dandruff conditions of the scalp. [Means for solving the problem]

[0014] The present disclosure is directed to compositions and methods for providing treatment for scalp disorders. More specifically, the present disclosure is directed to methods and compositions comprising Bacillus velezensis fermentation products and / or metabolites thereof for treating scalp disorders, including dandruff.

[0015] The present inventors unexpectedly observed that the fermentation product (culture supernatant) of Bacillus velezensis not only reduced the growth of Staphylococcus spp. and Malassezia spp., but also eliminated biofilms of Malassezia spp. and Staphylococcus spp.

[0016] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis fermentate and / or a metabolite thereof, the skin care composition alleviating and / or treating said scalp disorder.

[0017] In one aspect, the scalp disorder is selected from the group consisting of excessive scalp dandruff (seborrheic dermatitis), scalp ecoflora imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, or any one combination thereof.

[0018] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof, which alleviates and / or treats said scalp disorder and reduces the growth of Staphylococcus species.

[0019] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment and / or metabolites thereof, which alleviates and / or treats said scalp disorder and reduces the proliferation of Malassezia spp.

[0020] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof, which reduces and / or treats said scalp disorder and removes biofilms of Malassezia and Staphylococcus species.

[0021] In one embodiment, the composition is a skin care product comprising an effective amount of a skin care composition for use in treating a scalp disorder and one or more dermatologically or skin care acceptable components, said skin care composition comprising an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof, said skin care product alleviating and / or treating said scalp disorder.

[0022] In one embodiment, the composition is a skin care product selected from the group consisting of a lotion, serum, jelly, cream, gel, emulsion, mask, patch or stick comprising one or more dermatologically or skin care acceptable components and at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% up to 10% of a skin care composition by weight of the total skin care formulation (including an effective amount of Bacillus velezensis fermentate and / or metabolites thereof) described herein.

[0023] In some embodiments provided herein, the composition comprises an effective amount of a Bacillus velezensis fermentate, and / or a Bacillus velezensis cell lysate, and / or a Bacillus velezensis fragment, and / or a Bacillus velezensis metabolite for treating a scalp disorder, wherein the Bacillus velezensis is a bacterial strain having a 16S ribosomal RNA sequence exhibiting at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469. a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147473, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147471, and any one combination thereof.

[0024] In one embodiment, the method is a method of treating a scalp disorder in a subject in need thereof, comprising administering to the subject an effective amount of a Bacillus velezensis fermentation product and / or a metabolite thereof.

[0025] In one embodiment, the method is for treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject an effective amount of a Bacillus velezensis fermentation product and / or a metabolite thereof.

[0026] In some embodiments provided herein, the method comprises administering an effective amount of a Bacillus velezensis fermentate, and / or a Bacillus velezensis cell lysate, and / or a Bacillus velezensis fragment, and / or a Bacillus velezensis metabolite for treating a scalp disorder, wherein the Bacillus velezensis is a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469. a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147473, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147471, and any one combination thereof.

[0027] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising topically administering to the subject a skin care product comprising a skin care composition as described herein.

[0028] Further provided herein are kits for treating skin conditions comprising the compositions described herein and instructions for use thereof, in some embodiments, the kits further comprise one or more applicators configured to apply the composition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The features and advantages of the present disclosure will be more readily understood by those skilled in the art upon reading the following detailed description. It should be understood that certain features of the present disclosure, which are described above and below in the context of separate embodiments for clarity, may also be provided in combination in a single element. Conversely, various features of the present disclosure, which are described in the context of a single embodiment for brevity, may also be provided separately or in any subcombination. It will be understood that, below, an embodiment referred to in the context of one broad aspect of the present invention may be equally applicable to each of the other broad aspects of the present invention described above. It will be further understood that, unless the context dictates otherwise, the embodiments described below may be combined.

[0030] Scalp disorders The term "scalp disorder" for the purposes of this disclosure includes excessive dandruff of the scalp (seborrheic dermatitis), imbalance of the scalp ecoflora, scalp discomfort, tinea versicolor, dry skin, skin irritation, or any combination thereof.

[0031] The term "dandruff condition" for the purposes of this disclosure refers to a condition that is manifested by excessive dryness of the scalp or excessive sebum secretion and may be characterized, in some cases, by the presence of dry or greasy or oily dandruff or, in addition, by the presence of itching and / or the presence of inflammation of the epidermis.

[0032] A dry, flaky condition indicates a xerosis of the scalp, which may be combined with an excessively rapid renewal of the stratum corneum. Dry dandruff flakes are generally in the form of small white or grey flakes that spread over the scalp and onto clothing, creating an unpleasant appearance.

[0033] The itchy, dry scalp can cause erythema, pruritus, or even inflammation.

[0034] A condition of excessive greasy or oily dandruff is a form of seborrheic dermatitis. A person suffering from seborrheic dermatitis has an erythematous scalp covered with large greasy or oily yellow scales that accumulate to form capsules. The scalp is itchy and often causes a burning sensation in the affected areas. This phenomenon can be amplified by the presence of pathogenic microorganisms, especially Malassezia species. These microorganisms, which have the property of releasing fatty acids from sebum, can damage the barrier function of the epidermis and cause inflammation.

[0035] During dandruff, the skin barrier becomes unbalanced, its integrity and hydration is compromised, and the ecoflora is disturbed: the scalp skin becomes irritated and itchy, brittle, dehydrated and prone to infection.

[0036] In addition to the yeast Malassesia species, Staphylococcus species such as S. aureus or S. epidermidis are associated with dandruff scalp, while Propionibacterial species are associated with healthy scalp (Saxena, et al., 2018, Front Cell Infect Microbiol, 8:346; Xu, et al. 2016, Sci Rep., 6:24877). Inhibiting certain populations of Staphylococcus may be beneficial in treating dandruff. Certain Staphylococcus species, including S. aureus, can form biofilms (Crit Rev Microbiol, 43:602-620, 2017).

[0037] Unexpectedly, the inventors have observed that the fermentation product (culture supernatant) of Bacillus velezensis can reduce the proliferation of Staphylococcus species and Malassezia species. Furthermore, the inventors have unexpectedly observed that the fermentation product (culture supernatant) of Bacillus velezensis can remove the biofilm of Malassezia species and Staphylococcus species, thereby reducing the dandruff condition of a subject in need thereof.

[0038] The Malassezia species described herein include, but are not limited to, Malassezia restricta (M. restricta), Malassezia globosa (M. globosa), Malassezia furfur (M. furfur), Malassezia sympodialis (M. sympodialis), Malassezia phylotype 5 (M. phylotype 5), other uncharacterized Malassezia species, and any combination thereof.

[0039] Staphylococcus species as described herein include, but are not limited to, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus capitis, other uncharacterized Staphylococcus species, and any combination thereof.

[0040] Microorganisms, their fragments, their cell lysates, their fermentation products and their metabolites As used herein, "microorganism" or "microbe" refers to bacteria, fungi, viruses, protozoa, and other microscopic organisms or microscopic organisms.

[0041] In some embodiments, Bacillus velezensis (B. velezensis) cells can be subjected to treatments to render them replication-defective, such as heat exposure, desiccation, gamma irradiation, or ultraviolet irradiation. Non-replicating B. velezensis can be dead cells or live cells that are no longer capable of cell division. Non-replicating B. velezensis can be intact cells or cells that have been subjected to partial or complete cell lysis. In some embodiments, non-replicating cells can include a mixture of intact cells and cell lysates.

[0042] As used herein, the terms "probiotic" or "probiotic microorganism", used interchangeably herein, refer to live microorganisms (including, e.g., bacteria or yeasts) that, when administered (topically or orally) in sufficient amounts, beneficially affect a host organism, i.e., by imparting one or more demonstrable benefits to the host organism, such as a reduction in a dandruff condition.

[0043] In one aspect, microorganisms suitable for use in the present invention include strains of Bacillus velezensis.

[0044] In some embodiments, a microorganism suitable for use in the present invention is a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 (SEQ ID NO: 6) deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147469, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 (SEQ ID NO: 7) deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473 ... The present invention includes bacterial strains having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 (SEQ ID NO: 8) deposited at the World Food Database (WFDB) under number CBS147471, and any one combination thereof.

[0045] The 16S ribosomal RNA sequence of Bacillus velezensis E04 is as follows: [ka]

[0046] The 16S ribosomal RNA sequence of Bacillus velezensis H02 is as follows: [ka]

[0047] The 16S ribosomal RNA sequence of Bacillus velezensis F03 is as follows: [ka]

[0048] As used herein, the term "fragment" or "fragment of Bacillus velezensis" or "fragment of at least one Bacillus velezensis microorganism" or "fragment thereof" refers more specifically to a fragment of said microorganism, by analogy with said complete microorganism, which exhibits efficacy in treating scalp dandruff conditions. Fragment of at least one Bacillus velezensis organism includes metabolites obtained from said Bacillus velezensis (also referred to as Bacillus velezensis metabolites). In one embodiment, the fragment of Bacillus velezensis includes one or more metabolites (active compounds, also referred to as bioactives) derived from the metabolism of the Bacillus velezensis microorganism, which also exhibit efficacy in treating scalp disorders.

[0049] As used herein, the terms "metabolite" or "metabolite of at least one Bacillus velezensis" or "metabolite thereof" or "Bacillus velezensis metabolite" or "active metabolite" or "biologically active substance" are used interchangeably and refer to any substance derived from the metabolism of a Bacillus velezensis microorganism and having efficacy in treating scalp disorders. In one aspect, the one or more metabolites are produced during the cultivation (fermentation) of at least one Bacillus velezensis for use in treating scalp disorders. Metabolites of Bacillus velezensis for use in the treatment of scalp disorders include, but are not limited to, primary metabolites (metabolites directly involved in normal growth, development and reproduction), soluble metabolites, fatty acids, sugars, sugar alcohols, peptides, proteins, nucleotides, secondary metabolites, polynucleotides and polysaccharides.

[0050] It will be apparent that the fragments may be used directly in the compositions and formulations of the invention, or one or more of the biologically active substances (metabolites) may be isolated from the fragment by any suitable means prior to use.

[0051] In one aspect, Bacillus velezensis metabolites and / or fragments suitable for use in the present invention may be administered in the form of a lysate.

[0052] As used herein, the term "cell lysate" or "lysate" refers to cells that have been lysed by any suitable means. The term "cell lysate" or "lysate" traditionally refers to a material obtained by disrupting or decomposing biological cells through a phenomenon known as cell lysis, thus releasing the intracellular biological components naturally contained within the cells of the microorganism of interest. For the purposes of the present disclosure, the term "lysate" is used without preference to indicate only whole cell lysates or fragments thereof obtained through the lysis of the microorganism of interest. The lysates used are thus formed in whole or in part from intracellular biological components as well as from components of the cell wall and cell membrane. Advantageously, the lysates used in the present invention may be whole cell lysates obtained through the lysis of the microorganism of interest. This cell lysis can be achieved by any suitable means, such as, but not limited to, osmotic shock, heat shock, ultrasonication, sonication, homogenization, shearing, chemical lysis or mechanical stress of the centrifugation type.

[0053] In one embodiment, the cell lysate of Bacillus velezensis contains one or more metabolites (also referred to as bioactives), active compounds derived from the metabolism of the Bacillus velezensis microorganism, which also exhibit efficacy in treating scalp disorders.

[0054] It will be apparent that the cell lysate may be used directly in the compositions and formulations of the invention or one or more of the biologically active substances (metabolites) may be isolated from the cell lysate by any suitable means prior to use.

[0055] The lysate can be used in various forms, in the form of a solution or in the form of a powder. The microorganisms can be included in the composition according to the invention in live, semi-active or inactivated or dead form.

[0056] "Inactivated" or "dead" microorganisms within the meaning of the present invention are microorganisms that are no longer capable of forming colonies in culture. Dead or inactivated microorganisms may have intact or disrupted cell membranes. Dead or inactivated microorganisms may be obtained by any method known to the skilled artisan.

[0057] In some embodiments, cell debris is removed prior to use. In one embodiment, the cell lysate is filtered prior to use. In one embodiment, the cells are lysed, for example, by sonication, homogenization, shearing, or chemical lysis.

[0058] As used herein, the term "fermentate" is to be understood as a composition in which one or more live microbial strains are grown in a nutrient medium. In one aspect, "fermentate" refers to the cell-free supernatant of a cell culture of at least one Bacillus velezensis strain. In one embodiment, the cells are removed by centrifugation, optionally followed by filter sterilization. In one embodiment, the fermentate (cell culture supernatant) is obtained by filtering the culture medium in which Bacillus velezensis cells have been cultured.

[0059] In one aspect, "fermentate" refers to a supernatant of a cell culture of Bacillus velezensis selected from the group of a fermentate of B. velezensis E04, a fermentate of B. velezensis H02, a fermentate of B. velezensis F03, or any one combination thereof.

[0060] In one embodiment, the fermentation product of at least one Bacillus velezensis strain contains one or more metabolites (active compounds, also called bioactives) derived from the metabolism of the Bacillus velezensis strain, which also exhibit efficacy in treating scalp disorders.

[0061] It will be apparent that the fermentate may be used directly in the compositions and formulations of the present invention or one or more of the biologically active substances (metabolites) may be isolated from the fermentate by any suitable means prior to use.

[0062] In one aspect, the fermentation product can include one or more bioactive substances having a molecular weight of less than 50 kDa produced during the fermentation of at least one Bacillus velezensis strain, wherein the bioactive substances reduce the growth of Malassezia species.

[0063] In one aspect, the fermentation product can include one or more bioactive substances having a molecular weight of less than 30 kDa produced during fermentation of at least one Bacillus velezensis strain, wherein the bioactive substances reduce the growth of Malassezia species.

[0064] In one aspect, the fermentation product can include one or more bioactive substances having a molecular weight of less than 10 kDa produced during fermentation of at least one Bacillus velezensis strain, wherein the bioactive substances reduce the growth of Malassezia species.

[0065] In one aspect, the fermentation can include one or more bioactive substances having a molecular weight of less than 3 kDa produced during fermentation of at least one Bacillus velezensis strain, wherein the bioactive substances reduce the growth of Malassezia species.

[0066] In one aspect, the fermentation product can include one or more metabolites, such as, but not limited to, soluble metabolites produced during fermentation of at least one Bacillus velezensis strain.

[0067] In one embodiment, a fermentate derived from the cultivation (fermentation) of Bacillus velezensis may be used in the methods and / or uses of the present invention.

[0068] The nutrient medium used for the preparation of the fermentate is any medium containing the necessary nutrients suitable for the growth of the selected microorganism. Suitable nutrients include, but are not limited to, aminopeptides, peptides, yeast extract and / or vitamins. The medium can be based on dairy products such as milk, cereals, fruits and / or vegetables.

[0069] As used herein, the term "soluble metabolite" refers to one or more metabolites present in the supernatant of a cell culture from which the cells have been removed. In one embodiment, the culture is grown to a cell density of at least about OD600 of 0.5. In one embodiment, the cells are removed by centrifugation. In one embodiment, the supernatant is filtered. It will be apparent that the supernatant may be used directly in the formulation of the invention, or one or more of the metabolites may be isolated from the supernatant by any suitable means prior to use.

[0070] In some embodiments, the compositions of the invention can include a Bacillus velezensis fermentation product from which all or substantially all of the Bacillus velezensis cells have been removed. Methods for separating cells from growth media are well known in the art and can utilize physical methods, such as centrifugation to produce a cell pellet and culture supernatant, filtration, ultrafiltration, tangential flow filtration, dead end filtration, or reverse osmosis. Alternatively or additionally, the separation method can be ligand-based, for example, including an antibody that specifically binds to Bacillus velezensis. The antibody can be bound to a solid support, such as a magnetic bead.

[0071] In some embodiments, the compositions of the present invention include a Bacillus velezensis strain that is partially or substantially isolated from the growth and inactivated medium. The Bacillus velezensis strain can be non-replicative (e.g., inactivated, e.g., by heat treatment). The cells can be subjected to lyophilization, i.e., freeze-drying, under conditions that preserve the viability of the cells. Methods of lyophilization are well known in the art.

[0072] In one embodiment, the fermentation can include a Bacillus velezensis strain that consists essentially of nonviable cells (e.g., intact cells).

[0073] The term "consisting essentially of" in reference to a fermentation includes at least 90% of the Bacillus velezensis having the specified characteristic (e.g., intact nonviable cells). Preferably, at least 95% have the specified characteristic. Preferably, at least 97% have the specified characteristic. Preferably, at least 99% have the specified characteristic. In some embodiments, at least 100% have the specified characteristic.

[0074] As used herein, "cell-free fermentate" (synonymous with the term "fermentation supernatant" or "cell-free cell culture supernatant") means that the fermentate is substantially free of viable Bacillus velezensis cells.

[0075] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a fermentation supernatant derived from the fermentation of at least one Bacillus velezensis strain, said composition alleviating and / or treating said scalp disorder.

[0076] In one embodiment, the fermentate for use in the compositions and methods and / or uses of the invention may be substantially free of viable Bacillus velezensis cells, typically having 0 (or substantially 0) viable cells / mL of fermentate.

[0077] Skin care compositions and products for treating skin disorders such as dandruff conditions As used herein, the term "skin care composition" refers to a composition that includes at least one skin care benefit agent capable of providing a skin care benefit.

[0078] As used herein, the terms "skin care benefit agent" or "active agent" or "bioactive" are used interchangeably and refer to Bacillus velezensis fermentates, and / or Bacillus velezensis cell lysates, and / or Bacillus velezensis fragments, and / or Bacillus velezensis metabolites that can provide a skin care benefit.

[0079] In some embodiments provided herein, the skin care benefit agent comprises an effective amount of a Bacillus velezensis fermentate, and / or a Bacillus velezensis cell lysate, and / or a Bacillus velezensis fragment, and / or a Bacillus velezensis metabolite for treating a scalp disorder, wherein the Bacillus velezensis is a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 (SEQ ID NO: 6) deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 (SEQ ID NO: 7) deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147473, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 (SEQ ID NO: 8) deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147471, and any one combination thereof.

[0080] The term "skin care benefit" as used herein refers to the beneficial effect provided by the active agent (or skin care composition and / or skin care product comprising an effective amount of said active agent) when topically applied to skin. In one aspect of the present invention, the skin care benefit is selected from the group consisting of: prevention of excessive dandruff, relief of excessive dandruff, treatment of excessive dandruff, reduction of the occurrence of Malassezia species and / or Staphylococcus species on the skin (scalp), removal of biofilm formed by Malassezia species and / or Staphylococcus species on the skin (scalp), prevention or reduction of biofilm formation by Malassezia species and / or Staphylococcus species on the skin (scalp), improvement of skin barrier function, skin moisturization (protection of skin from dehydration by maintaining, restoring and / or enhancing skin moisturization), or any one combination thereof.

[0081] As used herein, the term "biofilm" refers to a community of microorganisms embedded in an extracellular polymeric matrix attached to a surface. The extracellular polymeric matrix is ​​a polymeric assembly generally composed of extracellular DNA, proteins and polysaccharides. A biofilm may have one or more microorganisms and may further contain water and other trapped particulate matter. The microorganisms may be gram-positive or gram-negative bacteria (aerobic or anaerobic); algae, protozoa and / or yeast or filamentous fungi. In one embodiment, the biofilm is a living cell comprising one or more Malassezia species.

[0082] As used herein, "surface" refers to any structure having sufficient mass to allow the attachment of a biofilm. Surfaces include hard and soft surfaces. Hard surfaces include, but are not limited to, metals, glass, ceramics, wood, minerals (rock, stone, marble, granite), aggregates such as concrete, plastics, composites, hard rubber materials, and gypsum. Other surfaces may be biological surfaces such as skin, scalp, or keratin.

[0083] Additional skin care benefit agents can include anti-dandruff actives.

[0084] Anti-dandruff actives include, for example, keratolytic agents such as salicylic acid and its various forms of sulfur, keratinization regulators such as zinc pyrithione, pyridinethione salts, trihalocarbamides, triclosan, azole compounds, antifungal polymers, allantoin, steroids such as topical corticosteroids, tar or polytar (coal tar), undecylenic acid, fumaric acid, allylamines and mixtures thereof, ciclopirox, octopirox, piroctone olamine, clobetasol propionate, betamethasone valerate, tea tree oil, thyme and catnip oil mixtures, topical antifungals such as selenium sulfide, imidazoles (e.g., ketoconazole), hydroxypyridones (e.g., ciclopirox), naturopathic agents such as Melaleuca seed oil, aloe vera and probiotic microorganisms. (Indian J. Dermatol,2010 Apr-Jun;55(2):130-134).

[0085] The Bacillus velezensis fermentate, and / or Bacillus velezensis cell lysate, and / or Bacillus velezensis fragments, and / or Bacillus velezensis metabolites skin care benefit agents further include agents (bioactives) that reduce the growth of Malassezia spp. and / or Staphylococcus spp. and agents that eliminate biofilms of Malassezia spp. and / or Staphylococcus spp.

[0086] In one aspect, the skin care benefit agent is a bioactive selected from the group consisting of a bioactive having a molecular weight of less than 50 kDa, a bioactive having a molecular weight of less than 30 kDa, a bioactive having a molecular weight of less than 10 kDa, a bioactive having a molecular weight of less than 3 kDa, and any combination thereof.

[0087] In one embodiment, the skin care benefit agent is a bioactive having a molecular weight of less than 50 kDa.

[0088] In one embodiment, the skin care benefit agent is a bioactive having a molecular weight of less than 30 kDa.

[0089] In one embodiment, the skin care benefit agent is a bioactive having a molecular weight of less than 10 kDa.

[0090] In one embodiment, the skin care benefit agent is a bioactive having a molecular weight of less than 3 kDa.

[0091] In one aspect, a skin care benefit agent (active agent) comprising a Bacillus velezensis fermentate and / or a fragment thereof is formulated into a skin care composition.

[0092] It will be appreciated that skin care compositions for use in the present invention may include at least one fermentation product of Bacillus velezensis and / or at least one metabolite of Bacillus velezensis.

[0093] Skin care compositions for use in accordance with the present invention may contain, for example, at least about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0% by weight of , about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 25.0%, about 30.0%, about 35.0%, about 40.0%, about 45.0%, or about 50.0% by weight of Bacillus velezensis fermentate and / or metabolites thereof.

[0094] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis fermentate and / or a metabolite thereof, the skin care composition alleviating and / or treating said scalp disorder.

[0095] In one aspect, the scalp disorder is selected from the group consisting of excessive scalp dandruff (seborrheic dermatitis), scalp ecoflora imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, or any one combination thereof.

[0096] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof, which alleviates and / or treats said scalp disorder and reduces the growth of Staphylococcus species.

[0097] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment and / or metabolites thereof, which alleviates and / or treats said scalp disorder and reduces the proliferation of Malassezia spp.

[0098] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof, which reduces and / or treats said scalp disorder and removes biofilms of Malassezia and Staphylococcus species.

[0099] In one embodiment, the composition is a skin care product comprising an effective amount of a skin care composition for use in treating a scalp disorder, the skin care composition comprising an effective amount of a Bacillus velezensis ferment and / or metabolites thereof, the skin care composition alleviating and / or treating said scalp disorder, and one or more dermatologically or skin care acceptable components.

[0100] In some embodiments provided herein, the skin care composition or product comprises an effective amount of a Bacillus velezensis fermentate, and / or a Bacillus velezensis cell lysate, and / or a Bacillus velezensis fragment, and / or a Bacillus velezensis metabolite for treating a scalp disorder, wherein the Bacillus velezensis is a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469. a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147473, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147471, and any one combination thereof.

[0101] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 50 kDa, the composition being a skin care composition that alleviates and / or treats the scalp disorder.

[0102] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 50 kDa, the composition reducing the growth of Malassezia species.

[0103] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 30 kDa, the composition being a skin care composition that alleviates and / or treats the scalp disorder.

[0104] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 30 kDa, the composition reducing the growth of Malassezia species.

[0105] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 10 kDa, the composition being a skin care composition that alleviates and / or treats the scalp disorder.

[0106] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 10 kDa, the composition reducing the growth of Malassezia species.

[0107] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 3 kDa, the composition being a skin care composition that alleviates and / or treats the scalp disorder.

[0108] In one embodiment, the composition is a skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 3 kDa, the composition reducing the growth of Malassezia species.

[0109] In one embodiment, the composition is a skin care product selected from the group consisting of a lotion, serum, jelly, cream, gel, emulsion, mask, patch or stick comprising one or more dermatologically or skin care acceptable components and at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% up to 10% of a skin care composition by weight of the total skin care formulation (including an effective amount of Bacillus velezensis fermentate and / or metabolites thereof) described herein.

[0110] In some embodiments, the compositions of the present invention can include a combination of Bacillus velezensis fermentation products and / or metabolites thereof and a carrier that is one or more dermatologically or skin care acceptable components.

[0111] Bacillus velezensis may be non-replicating (e.g., inactivated by heat treatment). Dosage may vary, but is approximately 10 2 ~about 10 12 cfu / g dry weight equivalent, e.g., 1×10 2 cfu / g, 5×10 2 cfu / g, 1×10 3 cfu / g, 5×10 3 cfu / g, 1×10 4 cfu / g, 5×10 4 cfu / g, 1×10 5 cfu / g, 5×10 5 cfu / g, 1×10 6 cfu / g, 5×10 6 cfu / g, 1×10 7 cfu / g, 5×10 7 cfu / g, 1×10 8 cfu / g, 5×10 8 cfu / g, 1×10 9 cfu / g, 5×10 9 cfu / g, 1×10 10 cfu / g, 5×10 10 cfu / g, 1×10 11 cfu / g, 5×10 11 cfu / g, 1×10 12 The range of cfu / g dry weight may be as follows:

[0112] In some embodiments, the Bacillus velezensis fermentate and / or its metabolites can be stabilized using conventional stabilization techniques before or after mixing with one or more dermatologically or skin care acceptable components.

[0113] In one embodiment, the skin care composition is formulated into a skin care product / formulation for administration to the skin.

[0114] It will be further appreciated that skin care compositions for use in the present invention may further comprise one or more probiotic bacteria in addition to active agents derived from Bacillus velezensis (such as Bacillus velezensis fermentates and / or Bacillus velezensis metabolites).

[0115] The skin care composition may include additional compounds selected from the group consisting of preservatives, pH adjusters, antioxidants and chelating agents.

[0116] Preservatives include, but are not limited to, parabens, sodium benzoate, potassium sorbate, phenylethyl alcohol, ethyl lauryl arginate (LAE), and any combination thereof.

[0117] pH adjusters include, but are not limited to, weak acids, strong acids, any compound capable of adjusting the pH, such as, but not limited to, citric acid, or any combination thereof.

[0118] The skin care compositions described herein or any effective amount of said skin care compositions can be used in the formulations and skin care products.

[0119] As used herein, "skin care product" refers to a product comprising an effective amount of the skin care composition described herein, including, but not limited to, a cosmetic product, an aqueous solution, an emulsion, a serum, a jelly, a patch, a lotion, a topical moisturizer, a cream, a paste, a scented balm, an ointment, a pomade, a gel, a liquid, a spray, a foam, a kit, or any one combination thereof.

[0120] In one embodiment, the skin care product is formulated for topical application to the skin / scalp.

[0121] In one embodiment, the composition is a skin care product comprising an effective amount of a skin care composition for use in treating a scalp disorder, the skin care composition comprising an effective amount of a Bacillus velezensis ferment and / or metabolites thereof, the skin care composition alleviating and / or treating said scalp disorder, and one or more dermatologically or skin care acceptable components.

[0122] In one embodiment, the skin care product is a lotion, serum, jelly, cream, gel, emulsion, mask, patch, or stick comprising one or more dermatologically or skin care acceptable components and at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and up to 10% by weight of a skin care formulation described herein based on the total weight of the skin care formulation.

[0123] In one embodiment, the skin care product is a product comprising an effective amount of Bacillus velezensis ferment and / or metabolites thereof, wherein the microorganism and / or skin care composition comprising said microorganism is formulated in at least one form selected from the group consisting of a gel, an emulsion, a hydrogel, a loose or compact powder, a suspension or solution, a spray solution, or any combination thereof.

[0124] 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, paints, bioadhesives or suspensions, and / or may be prepared to contain liposomes, micelles and / or microspheres. Formulations of this type may be used in conjunction with an occlusive covering to retain moisture within the formulation as it evaporates from the body surface during and after application of the formulation to the body surface.

[0125] 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).Components of this type of vehicle can include water, aqueous buffers, non-aqueous solvents (e.g., ethanol, isopropanol, benzyl alcohol, 2-(2-ethoxyethoxy)ethanol, propylene glycol, propylene glycol monolaurate, glycofurol, or glycerol), oils (e.g., mineral oils such as liquid paraffin, natural or synthetic triglycerides, or silicone oils such as dimethicone). In particular, depending on the nature of the formulation as well as its intended use and site of application, the dermatological vehicle employed may contain one or more components (e.g., components other than water, in the case where the formulation is an aqueous gel) selected from the following list: 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 copolymers); preservatives (e.g., benzyl alcohol, benzalkonium chloride, chlorhexidine, chlorbutol, benzoates, potassium sorbate, or EDTA or a salt thereof); and pH buffers (such as a mixture of dihydrogen phosphate and hydrogen phosphate or a mixture of citric acid and hydrogen phosphate).

[0126] Skin care products include liquid lotions (true solutions) that contain water as a solvent and water soluble additives (solutes) such as, but not limited to, active agents, fragrances, colorants, preservatives, pH adjusters, chelating agents, or any one combination thereof.

[0127] Skin care products include dispersions such as emulsions (including but not limited to liquid in liquid [water in oil W / O, O / W, W / O / W], suspensions [solid / liquid or liquid / solid], aerosols [liquid / gas or solid / gas], foams / mousses [gas / liquid or gas / emulsion or gas / solid]). Examples of oil in water [O / W] emulsions include, but are not limited to, a combination of an aqueous phase, an emulsifier, a fatty phase, and at least one additive. The aqueous phase can include water, a humectant, and a stabilizer (including but not limited to synthetic polymers, carbomers, natural polymers, xanthan gum, acacia gum, carrageenan, gellan, or any one combination thereof). Emulsifiers include, but are not limited to, anionic emulsifiers, cationic emulsifiers, nonionic emulsifiers, amphoteric emulsifiers, silicone emulsifiers, and self-emulsifiers. Fatty phases (lipophilic components) include, but are not limited to, waxes, butters, fatty acid esters, triglycerides, vegetable oils, mineral oils (paraffins), silicones and thickeners / oil gelling agents.Additives include, but are not limited to, preservatives, fragrances (mostly lipophilic), colorants, antioxidants, chelating agents, active agents, pH adjusters (citric acid, lactic acid, AHA), neutralizers / strong base agents such as NaOH, trimethylamine (to gel acrylic polymers) and powders.

[0128] Skin care products include aqueous gels that include an aqueous phase (including water, humectants, active agents), gelling agents (including but not limited to synthetic polymers, natural polymers, xanthan gum, acacia gum, carrageenan, gellan, etc.) and additives (including but not limited to fragrances, high HLB surfactants, colorants, active agents, preservative systems, pH adjusters, neutralizing agents, powders, etc.).

[0129] Skin care products include cleansing / surfactant systems (including but not limited to shampoos, shower gels, micellar waters, etc.) that contain an aqueous phase (water, humectants), surfactants, additives (including but not limited to fragrances, high HLB surfactants, colorants, actives, preservative systems, pH adjusters, neutralizers, powders, etc.) and optional gelling agents (including but not limited to synthetic polymers, natural polymers, xanthan gum, acacia gum, carrageenan, gellan, etc.).

[0130] As described herein, a skin care product or formulation comprising an effective amount of a Bacillus velezensis ferment and / or metabolites thereof as described herein provides a skin care benefit selected from the group consisting of: prevention of excessive dandruff, relief of excessive dandruff, treatment of excessive dandruff, reduction of occurrence of Malassezia spp. and / or Staphylococcus spp. on the skin / scalp, improvement of skin barrier function, skin moisturization (protection of the skin / scalp from dehydration by maintaining, restoring and / or enhancing skin moisturization), or any one combination thereof.

[0131] The skin care products (formulations) of the present invention may also incorporate a dermatologically or skin care acceptable carrier, which may be any carrier used in the art, including 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-based mixtures of such carriers.

[0132] As used herein, the terms "dermatologically acceptable" or "dermatologically acceptable carrier" or "skin care acceptable" or "skin care acceptable carrier" refer to compounds or compositions that may be incorporated into dermatological or skin care formulations without causing undesirable biological effects or undesirable interactions with other components of the formulation.

[0133] As used herein, "carrier" or "vehicle" refers to a carrier material suitable for incorporation into a topically applied composition. Carriers and vehicles useful herein include any material known in the art that is non-toxic and does not adversely interact with other components of the formulation in which it is contained.

[0134] The term "aqueous" refers to a formulation that contains water or that becomes water-containing after application to the skin or mucosal tissue.

[0135] The skin care products described herein may further comprise one or more known dermatologically or skin care acceptable components or other components that are effective for skin care use, provided that the optional components are physically and chemically compatible with the essential components described herein or do not otherwise unduly impair the stability, aesthetics or performance of the product. Non-limiting 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.

[0136] In one embodiment, the dermatologically or skincare acceptable component is a dermatologically acceptable carrier, comprising about 10% to about 99.9% by weight, alternatively about 50% to about 95% by weight, alternatively about 75% to about 95% by weight of a dermatologically acceptable carrier. Suitable carriers for use with the compositions include, for example, those used in formulating mousses, tonics, gels, and skin moisturizer lotions. Carriers include water; organic oils; silicones, such as volatile silicones, amino or non-amino silicone gums or oils, and mixtures thereof; mineral oils; vegetable oils, such as olive oil, castor oil, rapeseed oil, coconut oil, wheat germ oil, sweet almond oil, avocado oil, macadamia oil, apricot oil, safflower oil, kukui oil, camellia oil, calendula seed oil, lemon oil, and mixtures thereof; waxes; and organic compounds, such as C2-C 10 Alkanes, acetone, methyl ethyl ketone, volatile organic compounds C1-C 12 Alcohol, C1-C 20 Esters of acids and C1-C8 alcohols, such as methyl acetate, butyl acetate, ethyl acetate and isopropyl myristate, dimethoxyethane, diethoxyethane, C 10 ~C 30 Fatty alcohols, such as lauryl alcohol, cetyl alcohol, stearyl alcohol and behenyl alcohol; 10 ~C 30 Fatty acids, such as lauric acid and stearic acid; 10 ~C 30 Fatty amides, such as lauric acid diethanolamide; C 10 ~C 30 Fatty alkyl esters, such as C 10 ~C 30 The carrier may include fatty alkyl benzoates; hydroxypropyl cellulose; and mixtures thereof. In one embodiment, the carrier includes water, fatty alcohols, volatile organic alcohols, and mixtures thereof. Other carriers can be formulated by one skilled in the art.

[0137] The skin care products described herein may further comprise from about 0.1% to about 10%, alternatively from about 0.2% to about 5.0%, of a gelling agent which assists in imparting a desired viscosity to the composition. 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., EMA 81 available from Monsanto); non-neutralized crosslinked alkyl ether / acrylic acid copolymers (e.g., SALCARE™ SC90 available from Allied Colloids); non-neutralized crosslinked copolymers of sodium polyacrylate, mineral oil, and PEG-1 trideceth-6 (e.g., SALCARE™ SC91 available from Allied Colloids); non-neutralized crosslinked copolymers of methyl vinyl ether and maleic anhydride (e.g., STABILEZE™ QM-PVM / MA copolymer available from International Specialty Products); hydrophobically modified nonionic cellulose polymers; hydrophobically modified ethoxylated urethane polymers (e.g., UCARE™ Polyphobe, an alkali swellable polymer available from Union Carbide). Series); and combinations thereof. In this context, the term "non-neutralized" means that the optional polymer and copolymer gellant materials contain non-neutralized acid monomers.

[0138] The dermatologically or skincare acceptable medium may contain fatty substances, generally in a proportion of about 10 to about 90% by weight based on the total weight of the product, and the fatty phase contains at least one liquid, solid or semisolid fatty substance. Fatty substances include, but are not limited to, oils, waxes, gum-like substances and so-called pasty 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 skincare product may contain one or more conventional skincare or dermatological additives or adjuvants, such as, but 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).

[0139] The dermatologically acceptable carrier can be a moisturizing formulation containing at least one emulsifier, at least one surfactant, or any combination thereof.

[0140] In one embodiment, the skin care product is a product comprising a first skin care composition and a second skin care composition, the first skin care composition comprising an effective amount of a Bacillus velezensis ferment and / or metabolites thereof, and the second skin care composition comprising at least an effective amount of at least one second active agent (such as an anti-dandruff active, a skin conditioning agent, a skin care active ingredient material, etc.) for topical administration.

[0141] The skin care compositions and products may further comprise skin care active ingredients including sunscreens, moisturizers, humectants, skin benefit agents, adhesion promoters such as surfactants, occlusive agents, moisture barriers, lubricants, emollients, anti-aging agents, antistatic agents, abrasives, antimicrobial agents, conditioners, exfoliants, fragrances, viscosity agents, 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.

[0142] Other ingredients that may be included in the skin care composition or product include at least one active ingredient for treating or preventing a skin condition, providing a skin care benefit, or providing a moisturizing benefit to the skin, such as zinc oxide, petrolatum, white petrolatum, mineral oil, cod liver oil, lanolin, dimethicone, hard fat, vitamin A, allantoin, calamine, kaolin, glycerin, or colloidal oatmeal and combinations thereof, one or more natural moisturizing factors (e.g., ceramides, hyaluronic acid, glycerin, squalane, amino acids, cholesterol, fats, etc.), Examples of suitable lipid extracts include, but are not limited to, fatty acids, triglycerides, phospholipids, glycosphingolipids, urea, linoleic acid, glycosaminoglycans, mucopolysaccharides, sodium lactate or sodium pyrrolidone carboxylate), glycerides, apricot kernel oil, canola oil, squalane, squalene, coconut oil, corn oil, jojoba oil, jojoba wax, lecithin, olive oil, safflower oil, sesame oil, shea butter, soybean oil, sweet almond oil, sunflower oil, tea tree oil, shea butter, coconut oil, cholesterol, cholesterol esters, wax esters, fatty acids, and orange oil.

[0143] Any number of dermatologically acceptable materials commonly used in skin care products can also be incorporated into the present skin care products, such as skin conditioning agents and skin coloring agents.

[0144] Skin conditioning agents as defined herein include, but are not limited to, astringents that tighten the skin; exfoliants that remove dead skin cells; emollients that help maintain smooth, soft and supple appearance; humectants that increase the moisture content of the skin's surface layer; occlusive agents that retard the evaporation of water from the skin surface; and various compounds that enhance the appearance of dry or damaged skin, reduce flaking, and restore suppleness.Skin conditioning agents are well known in the art (see, for example, Green et al. (WO 01 / 07009)) and are commercially available from various sources. Suitable examples of skin conditioning agents include, but are not limited to, lactobionic acid, gluconic acid, alpha-hydroxy acids, beta-hydroxy acids, polyols, hyaluronic acid, D,L-panthenol, polysalicylates, vitamin A palmitate, vitamin E acetate, glycerin, sorbitol, silicones, silicone derivatives, lanolin, natural oils, xylitol, fucose, rhamnose, xylitol, betaine, and triglyceride esters. Skin conditioning agents include 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), and tartaric acid (CAS No. 133-37-9, Alfa Aesar). 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, Del.).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 WO 02 / 012530 by Bramucci.

[0145] The skin care compositions and products may contain skin care additives such as, but not limited to, colorants / dyes, fragrances, active agents, preservatives, pH adjusters, chelating agents, and antioxidants.

[0146] In one embodiment, the skin care product is a product comprising a first skin care composition and a second skin care composition, the first skin care composition comprising an effective amount of a Bacillus velezensis ferment and / or metabolites thereof, and the second skin care composition comprising at least an effective amount of at least one second active selected from anti-dandruff actives for topical administration. Anti-dandruff actives of this type include, for example, keratolytic agents such as salicylic acid and its various forms of sulfur, keratinization regulators such as zinc pyrithione, pyridinethione salts, trihalocarbamides, triclosan, azole compounds, antifungal polymers, allantoin, steroids such as topical corticosteroids, tar or polytar (coal tar), undecylenic acid, fumaric acid, allylamines and mixtures thereof, ciclopirox, octopirox, piroctone olamine, clobetasol propionate, betamethasone valerate, tea tree oil, thyme and catnip oil mixtures, topical antifungals such as selenium sulfide, imidazoles (e.g. ketoconazole), hydroxypyridones (e.g. ciclopirox), naturopathic agents such as Melaleuca seed oil, aloe vera and probiotic microorganisms. (Indian J. Dermatol,2010 Apr-Jun;55(2):130-134).

[0147] In one embodiment, the skin care product comprises a first skin care composition and a second skin care composition, wherein the first skin care composition comprises an effective amount of a Bacillus velezensis ferment and / or metabolites thereof; the second skin care composition comprises at least an effective amount of at least one second active agent selected from an anti-dandruff active agent for topical administration, and the first skin care composition is formulated in at least one form selected from the group consisting of a gel, an emulsion, a hydrogel, a loose or compact powder, a suspension or solution, or a spray solution.

[0148] The skin care compositions and skin care products described herein can also be part of a kit that provides one or more skin care benefits, such as, but not limited to, a kit for preventing or alleviating dandruff conditions.

[0149] In one aspect, the kit comprises an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof for the treatment of a dandruff condition, and instructions for administration to a subject in need thereof.

[0150] In one aspect, the kit comprises a skin care product for treating a dandruff condition in a subject in need thereof, the skin care product comprising an effective amount of a Bacillus velezensis ferment and / or metabolites thereof and instructions for administering the skin care product to a subject in need thereof.

[0151] Method for the Microbial Treatment of Scalp Disorders - Patent application The skin care compositions and products described herein can be used in methods for treating scalp disorders.

[0152] Further provided herein is a method of treating a scalp disorder in a subject in need thereof, comprising administering to the subject an effective amount of a Bacillus velezensis fermentation product and / or a metabolite thereof.

[0153] In one aspect, the scalp disorder is selected from the group consisting of excessive scalp dandruff (seborrheic dermatitis), scalp ecoflora imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, or any combination thereof.

[0154] In one embodiment, the Bacillus velezensis ferment is administered topically.

[0155] In one aspect, the Bacillus velezensis fermentation is a fermentation of at least one Bacillus velezensis strain selected from the group consisting of B. velezensis E04, B. velezensis H02, B. velezensis F03, and any combination thereof.

[0156] In one embodiment, the method is a method of treating a scalp disorder in a subject in need thereof, comprising administering to the subject an effective amount of a Bacillus velezensis fermentation product and / or a metabolite thereof.

[0157] In one embodiment, the method is a method of treating a scalp disorder in a subject in need thereof, comprising administering an effective amount of a composition comprising a Bacillus velezensis fermentation product and / or a metabolite thereof, wherein the composition alleviates and / or treats the scalp disorder.

[0158] A method for treating and / or alleviating a dandruff condition. The skin care compositions and products described herein can be used in methods for treating dandruff conditions.

[0159] Further provided herein is a method of treating a dandruff condition in a subject in need thereof, comprising administering to the subject an effective amount of a Bacillus velezensis fermentate and / or a metabolite thereof.

[0160] In one embodiment, the method is for treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject an effective amount of a Bacillus velezensis fermentation product and / or a metabolite thereof.

[0161] In some embodiments provided herein, the method comprises administering an effective amount of a Bacillus velezensis fermentate, and / or a Bacillus velezensis cell lysate, and / or a Bacillus velezensis fragment, and / or a Bacillus velezensis metabolite for treating a scalp disorder, wherein the Bacillus velezensis is a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469. a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147473, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147471, and any one combination thereof.

[0162] In one embodiment, the Bacillus velezensis ferment is administered topically.

[0163] In one aspect, the Bacillus velezensis fermentation is a fermentation of at least one Bacillus velezensis strain selected from the group consisting of B. velezensis E04, B. velezensis H02, B. velezensis F03, and any combination thereof.

[0164] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising topically administering to the subject a skin care product comprising a skin care composition as described herein.

[0165] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof, wherein the Bacillus velezensis fermentate and / or metabolites thereof are formulated into a single composition, and the composition is administered to the skin or scalp of the subject.

[0166] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof comprising administering to the subject an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof, wherein the Bacillus velezensis fermentate and / or metabolites thereof are formulated into a single composition, the composition further comprising a compound selected from the group consisting of skin care excipients, butyric acid, glucose, glycogen, magnesium ascorbyl phosphate, cetyl alcohol, dimethicone, isopropyl myristate, glycerol, propylene glycol, quaternium-52, ethanol, or any one combination thereof.

[0167] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering a skin care composition for use in treating a dandruff condition, wherein the skin care composition comprises an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof, and wherein the composition alleviates and / or treats the dandruff condition.

[0168] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a skin care product for use in treating a dandruff condition, wherein the skin care product comprises a skin care composition comprising an effective amount of a Bacillus velezensis ferment and / or metabolites thereof, wherein the composition alleviates and / or treats the dandruff condition.

[0169] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis fermentate, wherein the Bacillus velezensis fermentate comprises one or more bioactive substances having a molecular weight of less than 50 kDa, and wherein the composition reduces and / or treats the scalp disorder.

[0170] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 50 kDa, and the composition reducing the proliferation of Malassezia species.

[0171] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis fermentate, wherein the Bacillus velezensis fermentate comprises one or more bioactive substances having a molecular weight of less than 30 kDa, and wherein the composition reduces and / or treats the scalp disorder.

[0172] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 30 kDa, and the composition reducing the proliferation of Malassezia species.

[0173] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis fermentate, wherein the Bacillus velezensis fermentate comprises one or more bioactive substances having a molecular weight of less than 10 kDa, and wherein the composition alleviates and / or treats the scalp disorder.

[0174] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 10 kDa, and the composition reduces the proliferation of Malassezia species.

[0175] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis fermentate, the Bacillus velezensis fermentate comprising one or more bioactive substances having a molecular weight of less than 3 kDa, and the composition alleviates and / or treats the scalp disorder.

[0176] In one embodiment, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 3 kDa, and the composition reduces the proliferation of Malassezia species.

[0177] In one embodiment, the method is a method of treating and / or alleviating a scalp dandruff condition in a subject in need thereof, comprising administering to the subject a skin care product for use in treating a dandruff condition, wherein the skin care product comprises a skin care composition comprising an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof, wherein the Bacillus velezensis fermentate and / or metabolites thereof reduce the proliferation of Staphylococcus and / or Malassezia species.

[0178] In one embodiment, the method is a method of treating and / or alleviating a scalp dandruff condition in a subject in need thereof, comprising administering to the subject a skin care product for use in treating a dandruff condition, wherein the skin care product comprises a skin care composition comprising an effective amount of a Bacillus velezensis fermentate and / or metabolites thereof, wherein the Bacillus velezensis fermentate and / or metabolites thereof remove biofilms of Malassezia spp. and / or Staphylococcus spp.

[0179] In one aspect, the method is a method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising topically administering a skin care product comprising a skin care composition described herein.

[0180] In one aspect, the method is a method of treating and / or alleviating an excessive dandruff condition of the scalp in a subject in need thereof, comprising topically administering a skin care product comprising a skin care composition as described herein, wherein the skin care product is selected from the group consisting of lotions, serums, jellies, creams, gels, emulsions, solid cosmetics, masks, patches, and sticks comprising at least 1%, 2%, 3%, 4% to up to 5% of the skin care composition by weight based on the total weight of the skin care product.

[0181] In one embodiment, the method is for treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject an effective amount of a Bacillus velezensis fermentation product and / or a metabolite thereof.

[0182] General definition The disclosures of all cited patent and non-patent publications are incorporated herein by reference in their entirety.

[0183] In this disclosure, a number of terms and abbreviations are used. The following definitions apply unless specifically stated otherwise.

[0184] As used herein, the articles "a," "an," and "the" preceding an element or component of the invention are intended to be open ended with regard to the number of instances (i.e., occurrences) of that element or component. Thus, "a," "an," and "the" should be read to include one or at least one, and the singular form of an element or component also includes the plural, unless the number is specifically implied to be singular.

[0185] When an amount, concentration, or other value or parameter is given as a range, a preferred range, or a list of a higher preferred value and a lower preferred value, this should be understood to specifically disclose any range formed by any pair of any range upper limit or higher preferred value and any range lower limit or lower preferred value, regardless of whether the range is separately disclosed. When a range of numerical values ​​is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range. It is not intended that the scope of the invention be limited to the specific values ​​recited when defining a range.

[0186] The use of numerical values ​​within the various ranges set forth in this application is described as approximations, as if the term "about" preceded both the minimum and maximum values ​​within the stated range, unless expressly indicated otherwise. In this format, slight variations above and below the stated range can be used to achieve substantially the same results as values ​​within the range. The disclosure of these ranges also contemplates continuous ranges including each and every value between the minimum and maximum values. As used herein, the term "about" to modify the amount of an ingredient or reactant used refers to variations in the quantity that may occur, for example, through typical measuring and liquid handling procedures used to make concentrates or use solutions in the real world; through accidental errors in these procedures; through differences in the manufacture, source or purity of ingredients used to make the composition or carry out the method; and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for the composition resulting from a particular initial mixture. The claims include equivalents of quantities, whether or not modified by the term "about."

[0187] As used herein, the term "administer" or "administering" refers to the act of introducing one or more microorganisms (strains of microorganisms), skin care compositions, skin care formulations and / or skin care products to a subject in need of treatment for a scalp disorder.

[0188] Administration of one or more microorganisms (microbial strains), skin care compositions, skin care formulations, and / or skin care products to a subject includes applying or introducing one or more microorganisms (microbial strains), skin care compositions, skin care formulations, and / or skin care products to the scalp, skin surface, and skin cells in vitro or in vivo.

[0189] As used herein, the term "biological contaminant" refers to one or more unwanted and / or pathogenic biological entities, including, but not limited to, microorganisms, spores, viruses, prions, and mixtures thereof.

[0190] As used herein, the term "comprising" refers to the presence of a stated feature, integer, step, or component recited in the claim, but does not exclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. The term "comprising" is intended to include embodiments encompassed by the terms "consisting essentially of" and "consisting of." Similarly, the term "consisting essentially of" is intended to include embodiments encompassed by the term "consisting of."

[0191] As used herein, the terms "embodiment" or "disclosure" are not meant to be limiting and apply generally to any of the embodiments defined in the claims or described herein. These terms are used interchangeably herein.

[0192] As used herein, the term "excipient" refers to an inactive substance used as a carrier for an active ingredient in a formulation. An excipient may be used to stabilize an active ingredient in a formulation, such as the storage stability of the active ingredient. An excipient may also be used to bulk a formulation containing an active ingredient. "Active ingredients" include skin care benefit agents as described herein.

[0193] As used herein, the term "effective amount" refers to an amount sufficient to obtain a desired effect, such as the prevention, reduction and / or treatment of scalp disorders, such as the prevention, reduction and / or treatment of dandruff conditions.

[0194] As used herein, "prevent," "preventing," "prevention," and grammatical variations thereof refer to a method of partially or completely delaying or preventing the onset or recurrence of a disorder or condition (such as a scalp disorder) and / or one or more symptoms associated therewith, or a method of preventing a subject from acquiring or re-acquiring a disorder or condition, or a method of reducing the risk of a subject acquiring or re-acquiring a disorder or condition or one or more symptoms associated therewith.

[0195] As used herein, the terms "reducing," "reduce," and grammatical variations thereof, with respect to a particular feature, characteristic, feature, biological process, or phenomenon, refer to a decrease in that particular feature, characteristic, feature, biological process, or phenomenon, which may be decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or greater.

[0196] The terms "weight percent," "weight percentage (wt%)," and "weight-to-weight percentage (% w / w)" are used interchangeably herein. Weight percent refers to the percentage of a material based on its mass contained in a composition, mixture, solution, or product.

[0197] The term "16S rRNA" or "16S ribosomal RNA" refers to the rRNA that constitutes the small subunit of the prokaryotic ribosome. In bacteria, this sequence can be used to identify and characterize operational taxonomic units.

[0198] The term "ITS" or "internal transcribed spacer" refers to a region within a transcript that is excised and degraded during maturation by the ribosome. These sequences can be used for phylogenetic analysis and / or identification of fungi or yeasts.

[0199] The term moisturizer, lotion or body lotion refers to a low to medium viscosity emulsion of oil and water, most of which are oil-in-water type, but some are water-in-oil type, and the main effect in skin care applications is to hydrate the skin or reduce water loss from the skin. Almost all moisturizers contain a combination of emollients, occlusive agents and humectants. Emollients are primarily lipids and oils that hydrate and improve the appearance of the skin. A wide variety of suitable emollients are known and can be used herein (International Skin Care Ingredient Dictionary and Handbook, eds. Wenninger and McEwen, pp. 1656-61, 1626 and 1654-55 (The Skin care, Toiletry, and Fragrance Assoc., Washington, DC, 7th Edition, 1997) (referred to as the "ICI Handbook") contains many examples of suitable materials). Occlusive agents such as petrolatum, lanolin and beeswax create a hydrophobic barrier on the skin, thereby reducing transepidermal water loss. 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 contain emulsifiers to maintain emulsion stability, and thickeners can be used to achieve 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 other consumer-pleasing attributes.

[0200] The term "percent (%) sequence identity" or "percent (%) sequence similarity" as used herein with respect to a reference sequence is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the residues in the reference polynucleotide sequence, after optimal alignment of the sequences, inserting gaps, if necessary, to maximize the percent sequence identity.

[0201] As used herein, a microbial "strain" refers to a microorganism (such as a bacterium or fungus) that remains genetically unchanged as it grows or multiplies. Varieties of the same microorganism are encompassed.

[0202] As used herein, the term "biologically pure strain" means a strain that does not contain other microbial strains in sufficient amounts to prevent the strain from replicating or being detected by conventional techniques. "Isolated," when used in reference to the organisms and cultures described herein, encompasses not only biologically pure strains, but also any culture of an organism that is grown or maintained differently from that found in nature.

[0203] In one aspect, the skin cells described herein are mammalian skin cells, such as human skin cells or animal skin cells.

[0204] The term "sequence identity" or "sequence similarity" as used herein means that two polynucleotide sequences (i.e., candidate and reference sequences) are identical (i.e., 100% sequence identity) or similar (i.e., similar on a nucleotide-by-nucleotide basis) over the entire length of the candidate sequence. In comparing a candidate sequence to a reference sequence, when these two sequences are optimally aligned, the candidate sequence may contain additions or deletions (i.e., gaps) compared to the reference sequence (which contains no additions or deletions). Optimal alignment of sequences to determine sequence identity may be performed using any publicly available local alignment algorithm known in the art (e.g., ALIGN or Megalign (DNASTAR)), or by inspection.

[0205] It is intended that every numerical upper limit given throughout this specification will include every lower numerical limit, as if such lower numerical limit were expressly written herein. Every numerical lower limit given throughout this specification will include every higher numerical limit, as if such higher numerical limit were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0206] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0207] Non-limiting examples of the compositions and methods disclosed herein include the following. 1. A skin care composition for use in the treatment of a scalp disorder, comprising an effective amount of a Bacillus velezensis fermentation product and / or a metabolite thereof, said skin care composition alleviating and / or treating said scalp disorder. 1b. Bacillus velezensis refers to a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147469, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473 ... 2. The skin care composition of embodiment 1, wherein the bacterial strain is selected from the group consisting of a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the World Food Database (WFDB) under number CBS147471, and any one combination thereof. 2. The skin care composition of embodiment 1, wherein the scalp disorder is selected from the group consisting of excessive dandruff of the scalp (seborrheic dermatitis), imbalance of the scalp ecoflora, scalp discomfort, tinea versicolor, dry skin, skin irritation, or any combination thereof. 3. The skin care composition of embodiment 1, further comprising one or more anti-dandruff actives. 4. The skin care composition of embodiment 1, which reduces the growth of Staphylococcus and / or Malassezia species. 5. The skin care composition of embodiment 4, which reduces the growth of Staphylococcus species selected from the group consisting of Staphylococcus aureus and Staphylococcus epidermidis. 6. The skin care composition of embodiment 1, which removes biofilms of Malassezia spp. and / or Staphylococcus spp. 7. The skin care composition of embodiment 6, which removes biofilms of Malassezia and / or Staphylococcus species selected from the group consisting of Malassezia globosa and Staphylococcus aureus. 7b. The skin care composition of embodiment 4, which reduces the proliferation of Malassezia species selected from the group consisting of Malassezia globosa, Malassezia restricta, Malassezia sympodialis, Malassezia furfur, Malassezia phylotype 5, other uncharacterized Malassezia species, and any combination thereof. 8. The skin care composition of embodiment 1, further comprising an additional compound selected from the group consisting of excipients, preservatives, and pH adjusters. 8b. A skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 50 kDa, the composition alleviating and / or treating the scalp disorder. 8c. A skin care composition for use in the treatment of scalp disorders comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 50 kDa, the composition reducing the growth of Malassezia species. 8d. A skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 30 kDa, the composition alleviating and / or treating the scalp disorder. 8e. A skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 30 kDa, the composition reducing the growth of Malassezia species. 8f. A skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 10 kDa, the composition alleviating and / or treating the scalp disorder. 8g. A skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 10 kDa, the composition reducing the growth of Malassezia species. 8h. A skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 3 kDa, the composition alleviating and / or treating the scalp disorder. 8i. A skin care composition for use in the treatment of a scalp disorder comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 3 kDa, the composition reducing the growth of Malassezia species. 8j. Bacillus velezensis is a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147469, a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, The skin care composition of any one of embodiments 1-8i, wherein the bacterial strain is selected from the group consisting of a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the World Food Database (WFDB) under number CBS147471, and any one combination thereof. 9. Use of an effective amount of a skin care composition according to any one of embodiments 1 to 8j in a skin care product. 10. A skin care product comprising an effective amount of the skin care composition of any one of embodiments 1-9 and one or more dermatologically or skin care acceptable components. 11. The skin care product of embodiment 10, wherein the effective amount of skin care composition is at least about 1%, 2%, 3%, 4% to up to 5% by weight based on the total weight of the skin care product. 12. The skin care product of embodiment 10, which is formulated for topical administration. 12b. A skin care product comprising a first skin care composition and a second skin care composition, the first skin care composition comprising an effective amount of a first active agent consisting of a Bacillus velezensis ferment and / or metabolites thereof; and the second skin care composition comprising at least an effective amount of at least one second active agent selected from an anti-dandruff active agent, the second skin care composition being for topical administration. 12c. The skin care product of embodiment 12b, wherein the first skin care composition is formulated into at least one form selected from the group consisting of a gel, an emulsion, a hydrogel, a loose or compact powder, a suspension or solution, or a spray solution. 12d. The skin care product of embodiment 12b, wherein the at least one second composition comprises at least one member selected from the group consisting of hair lotions, shampoos, hair conditioners, detanglers, hair creams or gels, hair styling sprays, hair setting lotions, treatment lotions, dye compositions, hair repair lotions, permanent wave compositions, anti-hair loss lotions or gels, anti-parasitic or medicated shampoos, and scalp care products. 12e. Bacillus velezensis is a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147469, a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, The skin care product of embodiment 12, 12b, 12c or 12d, wherein the bacterial strain is selected from the group consisting of a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the World Food Database (WFDB) under number CBS147471, and any one combination thereof. 13. A method for treating a scalp disorder in a subject in need thereof, comprising administering to the subject an effective amount of a Bacillus velezensis fermentation product and / or a metabolite thereof. 14. The method of embodiment 13, wherein the scalp disorder is selected from the group consisting of excessive scalp dandruff (seborrheic dermatitis), scalp ecoflora imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, or any combination thereof. 15. The method of embodiment 13, wherein the fermentation product is administered topically. 16. A method for treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject an effective amount of a Bacillus velezensis fermentation product and / or a metabolite thereof. 16b. A method for treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 50 kDa, and the composition alleviates and / or treats the scalp disorder. 16c. A method for treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 50 kDa, and the composition reducing the growth of Malassezia species. 16d. A method for treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 30 kDa, and the composition alleviates and / or treats the scalp disorder. 16e. A method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 30 kDa, and the composition reducing the growth of Malassezia species. 16f. A method for treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 10 kDa, and the composition alleviates and / or treats the scalp disorder. 16g. A method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 10 kDa, and the composition reducing the proliferation of Malassezia species. 16h. A method for treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 3 kDa, and the composition alleviates and / or treats the scalp disorder. 16i A method of treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of a Bacillus velezensis ferment, the Bacillus velezensis ferment comprising one or more bioactive substances having a molecular weight of less than 3 kDa, and the composition reducing the proliferation of Malassezia species. 17. The method according to embodiments 16 to 16i, wherein the fermentation product and / or its metabolites are administered locally. 18. The method according to any one of embodiments 16 to 16i, wherein the fermentation products and / or metabolites are combined in a single composition. 19. The method according to embodiments 16-16i, wherein the composition is administered to the skin or scalp of the subject. 20. The method of embodiment 16, wherein the fermentate and / or its metabolites reduce the proliferation of Staphylococcus species and / or Malassezia species. 21. The fermentation products and / or their metabolites are capable of inhibiting Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus capitis, other uncharacterized Staphylococcus species, Malassezia globosa, Malassezia restricta, Malassezia sympodialis, Malassezia furfur, Malassezia phylotype 5, and / or their metabolites. 5), other uncharacterized Malassezia species, and any combination thereof. 22. The method of embodiment 16, wherein the fermentation product and / or its metabolites eliminate biofilms of Malassezia spp. and / or Staphylococcus spp. 22b. The method of embodiment 16, wherein the fermentate and / or its metabolites eliminate biofilms of Malassezia globosa and / or Staphylococcus aureus. 23. The method of any one of embodiments 13-22, wherein an effective amount of Bacillus velezensis fermentation product and / or metabolites thereof is administered as a skin care composition or product. 23b. Bacillus velezensis refers to a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147469, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473 ... The method according to any one of embodiments 13 to 23, wherein the bacterial strain is selected from the group consisting of a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the World Food Database (WFDB) under number CBS147471, and any combination thereof. 24. A method of treating and / or alleviating an excessive dandruff condition of the scalp in a subject in need thereof, comprising topically administering to the subject a skin care product comprising the skin care composition of any one of embodiments 1-8. 25. The method of embodiment 24, wherein the skin care product is selected from the group consisting of lotions, serums, jellies, creams, gels, emulsions, solid cosmetics, masks, patches and sticks comprising at least 1%, 2%, 3%, 4% and up to 5% of the skin care composition by weight based on the total weight of the skin care product. 26. A method for treating and / or alleviating a dandruff condition of the scalp in a subject in need thereof, comprising administering an effective amount of a first cosmetic active agent and an effective amount of at least one second cosmetic active agent, wherein the first cosmetic active agent comprises a Bacillus velezensis fermentate and / or a metabolite thereof. 27. The method of embodiment 26, wherein the first and second cosmetically active agents are administered to the skin or scalp of the subject. 28. The method of embodiment 26, wherein the first cosmetically active agent and the at least one second cosmetically active agent are combined in a single composition. 29. The method of embodiment 26, wherein the first cosmetically active agent and the at least one second cosmetically active agent are formulated in separate compositions. 30. The method of embodiment 26, wherein the at least one second cosmetic active agent comprises at least one anti-dandruff active agent. 31. The method of embodiment 30, wherein the at least one second cosmetic active agent comprises at least one member selected from the group consisting of zinc pyridinethione, salicylic acid, selenium disulfide, mixed oils of thyme and catnip, octopirox, probiotic microorganisms, and microorganisms. 32. The at least one second cosmetically active agent is a keratolytic agent, such as salicylic acid and its various forms of sulfur, a keratinization regulator, such as zinc pyrithione, pyridinethione salts, trihalocarbamides, triclosan, azole compounds, antifungal polymers, allantoin, a steroid, such as a topical corticosteroid, tar or polytar (coal tar), undecylenic acid, fumaric acid, allylamine and mixtures thereof, ciclopirox, octopirox, piroctone. The method of embodiment 31, comprising at least one member selected from the group consisting of olamine, clobetasol propionate, betamethasone valerate, tea tree oil, a mixture of thyme and catnip oil, topical antifungals such as selenium sulfide, imidazoles (e.g., ketoconazole), hydroxypyridones such as (e.g., ciclopirox), naturopathic agents such as Melaleuca seed oil, aloe vera, and probiotic microorganisms. 33. The method according to embodiment 32, wherein the flaky scalp condition is accompanied by dandruff, dry scalp, hyperseborrhoea of ​​the scalp, imbalance of the ecoflora, pruritus, inflammation of the scalp or imbalance of the scalp barrier function. 34. A method for preparing a cosmetic or dermatological composition for treating a dandruff condition, comprising combining an effective amount of a Bacillus velezensis ferment and / or metabolites thereof with at least one cosmetic or dermatological excipient. 35. A skin care composition for use in the treatment of a scalp disorder, comprising a fermentation product of Bacillus velezensis cells, said composition alleviating and / or treating said scalp disorder. 36. The skin care composition of embodiment 35, wherein all or substantially all of the Bacillus velezensis cells have been removed from the fermentation. 37. The composition of embodiment 36, wherein the fermentation product is a culture supernatant. 38. The composition of embodiment 36, further comprising one or more dermatologically or skin care acceptable components. 38b. Bacillus velezensis is a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147469, a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, 39. The skin care product of embodiment 35-38, wherein the bacterial strain is selected from the group consisting of a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the World Food Database (WFDB) under number CBS147471, and any one combination thereof. 39. A skin care product comprising the skin care composition of any one of embodiments 37-38. 40. Fermentation of Bacillus velezensis for use in the treatment of scalp disorders. 41. A fermentation product of Bacillus velezensis for use according to embodiment 40, wherein all or substantially all of the Bacillus velezensis cells have been removed from the fermentation product. 42. A fermentation product of Bacillus velezensis for use according to embodiment 40, wherein the fermentation product is a culture supernatant. 42b. Bacillus velezensis is a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147469, a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, a bacterial strain having a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, The fermentation product according to any one of embodiments 40 to 42, wherein the fermentation product is selected from the group consisting of bacterial strains having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the Waterford Foods Institute (WFDB) under number CBS147471, and any combination thereof. 43. A skin care product comprising the fermentation product according to any one of embodiments 40 to 42b. 44. A skin care composition for use in the treatment of a scalp disorder comprising a metabolite of a Bacillus velezensis cell, the metabolite being derived from the metabolism of a Bacillus velezensis microorganism and also exhibiting efficacy in the treatment of the scalp disorder, the composition alleviating and / or curing the scalp disorder. 45. The skin care composition of embodiment 39, further comprising one or more dermatologically or skin care acceptable components. 46. ​​A skin care product comprising the skin care composition of any one of embodiments 43-45. 47. A metabolite of Bacillus velezensis for use in the treatment of scalp disorders, said metabolite being derived from the metabolism of a Bacillus velezensis microorganism and also exhibiting efficacy in the treatment of said scalp disorders. 47b. Bacillus velezensis is a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147469, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, 48. The metabolite of embodiment 44 or 47, wherein the metabolite is selected from the group consisting of a bacterial strain having a 16S ribosomal RNA sequence that exhibits at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of B. velezensis F03 deposited at the Waters Food and Drug Administration (WFDB) under number CBS147471, and any one combination thereof. EXAMPLES

[0208] In the following examples, unless otherwise specified, parts and percentages are by weight and degrees are Celsius. It should be understood that this example shows an embodiment of the present disclosure, but is presented for illustrative purposes only. From the above discussion and this example, those skilled in the art can make various changes and modifications of the present disclosure to adapt the present disclosure to various usages and conditions. Such modifications are also intended to be included in the scope of the appended claims.

[0209] The following abbreviations used herein correspond to units of measurement, procedures, properties, or compounds as follows: "sec" or "s" means seconds, "min" means minutes, "h" or "hr" means hours, "μL" means microliter, "mL" means milliliter, "L" means liter, "mM" means millimolar, "M" means molar, "mmol" means millimole, "ppm" means parts per million, "wt" means weight, "wt%" means weight percent, "g" means gram, "mg" means milligram, "μg" means microgram, "ng" means nanogram, "conc." means concentration, and "Trt" means treatment.

[0210] Example 1 Isolation of microorganisms from rhizosphere soil A consortium of microorganisms from rhizosphere soil of a maize field was used to generate cell-free supernatants (fermentates). Growth media used in the isolation campaign included R2A, tryptic soy agar (TSA) and nutrient agar (NA). R2A medium consisted of casein acid hydrolysate (0.5 g / L), dextrose (0.5 g / L), dipotassium phosphate (0.3 g / L), magnesium sulfate (0.024 g / L), proteose peptone (0.5 g / L), sodium pyruvate (0.3 g / L), soluble starch (0.5 g / L) and yeast extract (0.5 g / L). Each liter of tryptic soy agar contained 17.0 g tryptone, 3 g soytone, 2.5 g glucose, 5 g sodium chloride, 2.5 g dipotassium phosphate and 15 g agar. NA medium has meat extract, 1 g / L, peptone, 5 g / L, sodium chloride, 5 g / L, yeast extract, 2 g / L, and agar, 15 g / L.

[0211] Plant roots were excavated by poking the soil with a shovel, and the root ball was cut and discarded above the plant mass above ground. Bulk soil was removed by manual shaking. After shaking, the soil in close contact with the roots was collected in a wash pan containing sterile water. The soil sample was suspended in sterile water. Up to 10 -4Serial dilutions of were plated on R2A, TSA and NA media. Agar plates were incubated at 28°C for 24-72 h. Colonies were picked, grown in liquid medium and stored at -80°C in 25% glycerol. Phylogenetic identity of the isolates was determined by sequencing the 16S rRNA region using primer set 8F (5'-AGA GTT TGA TYM TCC TGG CTC-3, SEQ ID NO:1) and 1492R (5'-CGG TTA CCT TGT TAC GAC TT-3, SEQ ID NO:2).

[0212] Example 2 Growth inhibition assays using isolated fermentates against bacterial targets To generate cell-free supernatants (also referred to as fermentates) from individual isolates described in Example 1, strains were revived from frozen stocks and grown in 1 mL TSB medium in deep-well microtiter plates. To prepare filtrates for microtiter plate assays, candidate strains were subcultured by inoculating 25 μL of reculture into 1100 μL of fresh TSB in 96 deep-well microtiter plates and grown for 3 days at 28° C. in a 75% RH chamber with shaking at 350 rpm. To harvest fermentates, cell cultures were centrifuged at >6,100 rcf at 4° C. Approximately 700 μL of supernatant was removed and filtered through a 0.2 μm filter with a 96-well filter plate.

[0213] As described herein, Staphylococcus species are associated with dandruff based on microbiome analysis of healthy and dandruff populations. For growth inhibition assays, Staphylococcus aureus (S. aureus) ATCC 6538 and Staphylococcus epidermidis (S. epidermidis) ATCC 49461 were selected as targets.

[0214] Inhibition assays with fermentations of different soil isolates were performed in a 384-well format. Fresh overnight shake flask cultures in TSB were diluted to an equivalent McFarland turbidity standard of 1.0 in 1x PBS (phosphate buffered saline). To prepare the starter culture stock used in the 384-well inhibition assay, this culture was then diluted again 1:500 in diluted TSB medium (50% TSB + 50% PBS (1x phosphate buffered saline). The 384-well inhibition assay was performed by adding 80 μl of starter culture to each well with 20 μL of test fermentation.

[0215] Fermentations from each isolate were evaluated for inhibition in replicate wells (n=4) within each 384-well plate. Sterile TSB medium was used as a negative control. The 384-well assay plates were incubated at 32° C. for 18 hours and growth in each well was measured by OD absorbance (600 nm) using a plate spectrophotometer.

[0216] Fermentations from three strains of Bacillus velezensis, namely Bacillus velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, Bacillus velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, and Bacillus velezensis F03 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147471 (Table 1), were identified as having efficacy based on the inhibition assay. As shown in Table 2, the fermentation products of these three strains had potent activity in inhibiting the growth of S. aureus and S. epidermidis species, both of which have been found to be associated with dandruff.

[0217] [Table 1]

[0218] [Table 2]

[0219] Example 3 Removal of Malassezia species biofilms by fermentation from Bacillus velezensis strains In this example, the yeast Malassezia globosa (M. globosa) ATCC MYA-4612 was used in a biofilm assay to evaluate fermentations from three strains of Bacillus velezensis: Bacillus velezensis E04, deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, Bacillus velezensis H02, deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS 147473, and Bacillus velezensis F03, deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147471. Yeast strains were routinely grown in MLN medium + palm oil at 32°C in a rotary shaker at a speed of 100 rpm. MLN medium consisted of 10 g / L bacteriological peptone, 2 g / L yeast extract, 8 g / L dried calf bile, 10 mL / L glycerol, 0.5 g / L glycerol monostearate and 5 mL / L Tween-60. Palm oil was supplemented at a concentration of 20 mL / L. After 3 days of incubation, 10 μL of fresh culture was inoculated into wells of a polystyrene 96-well plate containing 90 μL of MLN medium with palm oil as the carbon source. M. globosa was grown as sessile biofilm cells attached to the well walls of the microtiter plate by incubating the plate at 32°C for 48 hours without shaking.

[0220] To produce fermentations, the different strains were grown in TSB medium at 28° C. for 3 days in a shaking incubator with 75% RH and a shaking speed of 350 rpm using 1 mL of medium in a deep-well block. The cells were harvested by centrifugation using a table-top centrifuge at a speed of 4000 rpm, and the supernatant was filtered through a sterile 96-well 0.2 μm membrane filter unit before use (thus producing fermentations). To produce larger quantities of fermentations, selected strains were grown in 100 mL shaking flasks with 25 mL of TSB. The flasks were placed in a shaking incubator with a speed of 200 rpm for 2 days at 32° C.

[0221] For biofilm assays using microtiter plates, growth medium and unattached cells were removed, and wells were washed once with 1x phosphate-buffered saline solution (PBS) after biofilm formation. PBS is a mixture of pH-adjusted ultrapure grade phosphate buffer and saline, containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4 when diluted to a 1x working concentration. After washing, 150 μL of supernatants of bacterial strains were added to evaluate their effectiveness for biofilm removal. 150 μL of TSB was also added as a control. Biofilm plates were incubated at 32°C for 15 min without shaking to perform biofilm removal reactions. After incubation, the supernatant or control medium was removed. The amount of biofilm remaining in the wells was quantified by staining. Biofilm staining was performed by adding 150 μL of 0.1% crystal violet dissolved in water. Plates were incubated at room temperature for 3 min. After staining, 150 μL of 1× PBS was added to each well to remove unbound dye. This process was repeated once more. After washing, 150 μL of 70% ethanol was added to each well and incubated at room temperature for 5 min to release the dye. The dye intensity was measured at 570 nm using a microtiter plate reader. OD readings were used to quantify the amount of biofilm remaining after each treatment. Wells treated with TSB medium were used as a reference to calculate the biofilm removal rate.

[0222] After screening for Malassezia biofilm removal, fermentates from the soil isolates described in Example 1, the same three B. velezensis strains listed in Table 1, were also found to have activity in removing Malassezia biofilms. Results of activity for Malassezia biofilm removal using fermentates generated from flasks are shown in Table 3. Addition of fermentates from these strains resulted in a 77-80% reduction in biofilm compared to the TSB control. This experiment demonstrated the potential utility of supernatants from Bacillus velezensis strains for controlling Malassezia biofilms.

[0223] [Table 3]

[0224] Example 4 Removal of Staphylococcus aureus biofilms by Bacillus velezensis fermentation Staphylococcus aureus (S. aureus) ATCC 6538 was used in biofilm experiments to evaluate the efficacy of B. velezensis supernatants (fermentates) from three strains of Bacillus velezensis, namely Bacillus velezensis E04 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147469, Bacillus velezensis H02 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147473, and Bacillus velezensis H03 deposited at the Westerdijk Fungal Biodiversity Institute (WFDB) under number CBS147474. The efficacy of Staphylococcus velezensis F03 was evaluated. Tryptic soy broth medium (TSB) was routinely used for the growth of S. aureus. TSB (per liter) consisted of 17.0 g tryptone, 3.0 g soytone, 2.5 g glucose, 5.0 g sodium chloride, and 2.5 g dipotassium phosphate at pH 7.3. Biofilm formation medium (BFM) consisted of 50% BHI (brain heart infusion) and 50% YPD (yeast peptone dextrose).

[0225] B. velezensis strains were grown in TSB medium in deep-well blocks with 1 mL of medium for 3 days at 28°C for high throughput or in flasks with 25 mL of medium for 2 days at 32°C to obtain larger volumes. With deep-well blocks, the cultures were incubated in a shaker at a speed of 350 rpm. With flasks, the cultures were incubated in a shaker at a speed of 200 rpm. Supernatants (fermentates) were generated from the cultures after 48 hours of growth in a rotary shaker at a speed of 200 rpm. Cells were harvested by centrifugation at a speed of 4000 rpm using a table-top centrifuge, and the supernatant was filtered through a 0.2 μm membrane filter before use.

[0226] For the biofilm assay, 96-well sterile polystyrene microtiter plates were used. Ten microliters of a 2-hour culture of S. aureus from a fresh plate was transferred to each well containing 90 μL of biofilm formation medium (BFM). The plates were incubated at 37°C to allow biofilm formation on the walls of each well. After 24 hours of incubation, the growth medium was removed and 100 μL of 1X PBS was added to each well to gently wash and remove unbound cells. To test B. velezensis fermentation for S. aureus biofilm removal, 100 μL of supernatant was added to each well. As a control, 100 μL of TSB was used. For the biofilm removal reaction, the plates were incubated at 37°C for 20 minutes. After incubation, the solution in each well was removed and the wells were washed with 1X PBS. After emptied the PBS solution, the attached biofilm was stained with 100 μL of 0.1% crystal violet dye dissolved in water for 3 min at room temperature. Unbound dye was removed and each well was washed twice with 1× PBS. After emptied the PBS solution, 100 μL of 70% ethanol was added to suspend the dye bound to the biofilm. The dye intensity was measured at 570 nm using a spectrophotometer. OD was used to indicate the amount of biofilm remaining after treatment.

[0227] The biofilm removal results are shown in Table 4. Compared with the TSB control, the biofilm removal activity of S. aureus was more than 70% in the fermentations from these three strains.

[0228] [Table 4]

[0229] This result, combined with the results from Examples 2 and 3, demonstrated the unexpected properties of Bacillus velezensis strains in removing biofilms formed by Staphylococcus and Malassezia, two important species associated with dandruff and scalp disorders.

[0230] Example 5. Growth inhibition of Malassezia species by B. velezensis fermentates. The ability of B. velezensis fermentation to inhibit the growth of Malassezia species was evaluated. B. velezensis strain E04 was grown in Soyptone Urea Micronutrient (SUM) medium. Each liter of SUM medium (1.0x strength) consisted of 10 g soytone, 75 g glucose, 3 mL dipotassium phosphate (1M), 3.6 g urea, and 10 mL micronutrient stock. Each liter of micronutrient stock contained 1.47 g sodium citrate·2H2O, 1.47 g CaCl2·2H2O, 0.4 g FeSO4·7H2O, 0.1 g MnSO4·H2O, 0.1 g ZnSO4·H2O, 0.05 g CuCl2.2H2O, 0.1 g CoCl2·6H2O, and 0.1 g Na2MoO4·2H2O. SUM medium was adjusted to pH 7.3. Production of B. velezensis fermentations was carried out in 125 mL flat-bottom shake flasks containing 25 mL of medium. The flasks were placed in a platform shaker with a constant speed of 200 rpm at 32 °C. An overnight fresh seed culture was used as inoculum with a starting OD of 0.125 at 600 nm. After 48 hours of growth, the supernatant (fermentation liquid) was obtained by centrifugation and filter sterilization.

[0231] To determine the antifungal activity of B. velezensis fermentation products, growth inhibition assays were performed using Malassezia furfur (M. furfur) as the target. M. furfur ATCC 14521 was cultured from frozen stocks by inoculating 1:100 into mDixon medium containing 500 μg / mL chloramphenicol and incubated for 2 days at 32 °C in an orbital shaker at 250 rpm before antifungal susceptibility testing. Each liter of mDixon medium contains 36 g malt extract, 20 g dried oxol, 10 mL Tween-40, 6 g peptone, 2 mL glycerol, and 2 mL oleic acid. The pH of the medium was adjusted to 6. M. furfur cells were suspended in 1% Tween-80 to obtain an optical density equivalent to the turbidity standard McFarland 4.0. This standardized cell culture was once again diluted in a 1:10 ratio into Sabouraud-dextrose broth containing 500 μg / mL chloramphenicol supplemented with Tween-80 to 1%. Sabouraud-dextrose broth consists of dextrose (40 g / L) and peptone (10 g / L), the pH of which has been adjusted to 5.6.

[0232] For the growth inhibition assay, the final volume of the reaction was 600 μL, and the reactions were performed in 96 deep-well plates (Corning Costar 3960). First, a dilution series of the fermentation was generated by diluting the fermentation 2-fold, 4-fold, and 8-fold in phosphate buffered saline (PBS). PBS consists of 8 g NaCl, 0.2 g KCl, 1.42 g Na2HPO4, and 0.24 g KH2PO4 per liter. 150 μL of undiluted and diluted fermentation was dispensed into each well of the 96 deep-well plate. Then, 450 μL of a standardized culture of M. furfur in SabDex+Tween-80 was added to each well of the 96 deep-well plate containing different concentrations of the fermentation. The final concentration of fermentate in each reaction by volume was 25% for undiluted fermentate and 12.5%, 6.25% and 3.125% for diluted fermentate, respectively. The assay plates were incubated at 35°C and 75% relative humidity in an orbital shaker with an orbital throw of 25 mm and a shaking speed of 375 rpm. After 48 hours, optical density was measured at a wavelength of 600 nm in a microplate reader. Results were reported as % inhibition (Table 5). Percent inhibition was calculated as = 100 * (1 - (observed - min) / (max - min)), where observed = measured OD (average of test replicates), max = average OD of negative controls with PBS (no fermentate) and min = OD where no growth was observed (no M. furfur).

[0233] [Table 5]

[0234] As shown in Table 5, Bacillus velezensis fermentation was able to provide strong growth inhibition of Malassezia species. No significant differences were observed in the effect of B. velezensis fermentation on Malassezia inhibition (range 99.1-100%) when the Bacillus velezensis fermentation was derived from cultures grown at SUM 1.0x intensity, SUM 0.5x intensity or SUM 0.25x intensity.

[0235] Example 6 Fraction of fermentation of Bacillus velezensis To analyze the molecular weight of bioactive substances with antifungal activity, fermentations of B. velezensis were obtained from cells grown in 0.5x SUM medium as described in Example 5. Fractions of the fermentation were performed using EMD Amicon ultra-0.5 centrifugal filter units with molecular cutoffs of 10 kilodaltons, 30 kilodaltons and 50 kilodaltons (kDa). The flow-through was used in an antifungal assay using M. furfur as a target as described in Example 5. Table 6 shows the assay results using 3.125% fermentation. Up to 99% activity was seen in the 50 KDa, 30 KDa and 10 KDa flow-throughs, suggesting that the majority of the bioactive substances responsible for antifungal activity have a molecular weight of 10 kDa or less.

[0236] [Table 6]

Claims

1. A skin care composition for use in the treatment of a scalp disorder, comprising an effective amount of a Bacillus velezensis fermentate and / or a metabolite thereof, the skin care composition alleviating and / or treating said scalp disorder.

2. 2. The skin care composition of claim 1, wherein the scalp disorder is selected from the group consisting of excessive scalp dandruff (seborrheic dermatitis), scalp ecoflora imbalance, scalp discomfort, tinea versicolor, dry skin, skin irritation, or any combination thereof.

3. 10. The skin care composition of claim 1 further comprising one or more anti-dandruff actives.

4. 10. The skin care composition of claim 1, which reduces the growth of Staphylococcus and / or Malassezia species.

5. 10. The skin care composition of claim 1, which removes biofilms of Malassezia spp. and / or Staphylococcus spp.

6. 10. The skin care composition of claim 1, further comprising an additional compound selected from the group consisting of excipients, preservatives, and pH adjusters.

7. Use of an effective amount of the skin care composition according to any one of claims 1 to 6 in a skin care product.

8. A skin care product comprising an effective amount of the skin care composition of any one of claims 1 to 6 and one or more dermatologically or skin care acceptable components.

9. 9. The skin care product of claim 8, wherein the effective amount of the skin care composition is at least 1% and at most 5% by weight based on the total weight of the skin care product.

10. 10. The skin care product of claim 8 formulated for topical administration.

Images