Novel composition comprising malassezia postbiotics, method and use

Abstract

A topical composition, preferably a skincare and / or haircare composition and / or a composition to treat, care for or improve the appearance of the skin and / or scalp, more preferably an anti-dandruff composition and / or anti-acne composition, wherein such composition comprises one or more Malassezia postbiotics and wherein the Malassezia postbiotics are derived from Malassezia species furfur. A topical composition, preferably a cosmetic skincare composition for non-therapeutic use,wherein such composition comprises one or more Malassezia postbiotics and wherein the Malassezia postbiotics are derived from Malassezia species restricta, Malassezia species globosa or a mixture thereof; and preferably wherein the composition does not comprise Malassezin. A process for the preparation of a cosmetic composition, comprising the steps of: - Lysis of one or more Malassezia microorganisms, wherein the one or more Malassezia microorganisms comprise a CCL5 inhibitor and / or a CCL27 inhibitor and / or an IVL stimulator, to yield Malassezia postbiotics; and - Combining the Malassezia postbiotics with a cosmetically acceptable carrier and optionally one or more human milk oligosaccharides.

Description

[0001] DSM IP Assets B.V. 2024P00093WO 01

[0002] NOVEL COMPOSITION, METHOD AND USE

[0003] Field of the invention

[0004]

[0001] This invention relates to the fields of cosmetics such as skincare, haircare and suncare. More specifically this invention relates to a composition, method and use, for prevention and / or mitigation of (the signs of) inflammation and / or an inflammaging of a tissue, more specifically a keratinous tissue.

[0005] Background of the invention

[0006]

[0002] Inflammation, especially inflammation of a keratinous tissue such as the skin, is a biological response to injury and / or stress caused by external factors. The skin protects the body against exposure to such external factors, for example pollution, abrasions, UV radiation, and undesired microorganisms such as undesired bacteria, viruses, fungi and parasites.

[0007]

[0003] In response to any infection or injury, skin cells can produce several proteins to aid in combatting the infection or heal the injury. Produced cytokine proteins such as IL-4, IL-13 and TNF- alpha, involved in inflammatory processes, increase blood flow and vascular permeability in affected skin. As a result the skin becomes red and swollen. Over time, repeated and / or prolonged inflammatory responses can lead to structural damage of the skin resulting in increasing visible signs of aging such as wrinkles, sagging, and / or an uneven skin tone.

[0008]

[0004] Aging can affect the skin’s inflammatory processes and it is known in practice that older, thinner skin can be more prone to chronic low-grade inflammation. This phenomenon is also referred to as “inflammaging”, a combination of “inflammation” and “aging”. With increasing average age in populations across the world, also the importance and impact of skin inflammaging is increasing.

[0009]

[0005] In addition, also younger people can suffer from chronic, low-grade inflammation of the skin, for example in the form of psoriasis. Psoriasis is a chronic, inflammatory skin condition characterized by the rapid overproduction of skin cells, leading to the formation of thick, red, scaly patches on the skin's surface. Apart from the immediate effects, the skin may also become damaged for the longer term, making the skin look less attractive.

[0010]

[0006] Millions of people suffer from the condition psoriasis in a lighter or more severe form. Canavese et al. , in their article titled “Therapeutic Efficacy and Immunological Response of CCL5 Antagonists in Models of Contact Skin Reaction” , published online via PLoS ONE, vol. 5 (2010), item e8725, explain that psoriasis is an autoimmune disorder where immune cells are activated and accumulate in the skin. That is, the immune system attacks healthy skin cells, causing inflammation. According to Canavese et al. the chemokine CCL5 unfortunately plays a key role in directing these immune cells to the site of inflammation.

[0007] Hence, there is a need amongst consumers, old and young, to have access to solutions to reduce and / or mitigate damage from inflammation and / or inflammaging to the appearance of their skin.

[0011]

[0008] Pharmaceutical industry has developed products to tackle inflammation from a medical perspective. For example, the biological drug “secukinumab” was developed to neutralize IL-17A (lnterleukin-17A), a pro-inflammatory cytokine that plays a critical role in immune responses. Another developed biological drug is Adalimumab, a TNF-alpha inhibitor. The biological drug Ustekinumab was developed to block cytokines IL-12 and IL-23. However, all these drugs are administered via an injection and hence are not very desirable to customers in need of a cosmetic treatment.

[0012]

[0009] However, current cosmetic products merely address inflammation from a superficial perspective, focusing on soothing or masking of the visible signs.

[0013]

[0010] It would be an advantage to have a cosmetic solution that is able to also address, at least to some extent, the biological aspects of inflammation and / or inflammaging and / or to promote a more robust skin. Such a cosmetic solution could advantageously provide both relief from as well as longer term protection against a red and swollen skin associated with inflammation and / or inflammaging. As a result this may advantageously allow for an improved appearance of the skin.

[0011] Ro and Dawson describe in their article titled “The Role of Sebaceous Gland Activity and Scalp Microfloral Metabolism in the Etiology of Seborrheic Dermatitis and Dandruff" published in J Investig Dermatol Symp Proc vol. 10, 2005, pages 194 -197, that Malassezia yeasts, via their lipid metabolism, have a pathogenic role in the development of dry scalp flaking, also referred to as “dandruff’, and seborrheic dermatitis.

[0014]

[0012] On the other hand, US20190337927A1 describes Malassezia-derived compounds, including malassezin and indirubin, and / or chemical analogs thereof for modulating skin pigmentation and treating or preventing UV-induced skin damage, erythema, aging of the skin, sunburn, and hyperpigmentation in a subject. US 2017 / 0260133 A1 relates to malassezin and analogs thereof as skin brightening agents.

[0015]

[0013] Pagac et al., in their article titled “A New Generation of Postbiotics for Skin and Scalp: In Situ Production of Lipid Metabolites by Malassezia”, published in Microorganisms vol. 12, 2024, item 1711 , pages 1-17, describe that Malassezia species, such as Malassezia globosa, Malassezia restricta, Malassezia sympodialis, and Malassezia furfur, were able to produce bioactive lipid mediators that could be beneficial. However, unfortunately the (in-situ) production of such bioactive lipid mediators requires life Malassezia species to be present on the skin or scalp, which may be perceived as undesirable by customers. In addition, the life Malassezia organisms are fragile and not easy to handle when preparing a commercial product. This makes it challenging for industry to formulate the life Malassezia organisms into a consumer product.

[0016]

[0014] Aberg et al. in their article titled "Results of allergen-specific immunotherapy in atopic dogs with Malassezia hypersensitivity: a retrospective study of 16 cases" describes a retrospective study to report the clinical response and adverse effects of subcutaneous (i.e. under the skin) immunotherapy with Malassezia extract in dogs having atopic dermatitis. The dogs are treated with Malassezia extract in the form of Artuvetrin® 100 microgram / milliliter injections obtained from ArtuVet Animal Health BV.

[0017]

[0015] US2019 / 03451 describes the utilization of compounds produced by or derived from Malassezia yeast, including Malassezin, Indirubin, and chemical analogs thereof, as the basis for safe and efficacious skin brightening and skin darkening compositions.

[0018]

[0016] In view of the above, it remains desirable to provide a solution that could be appealing to cosmetic industry and to cosmetic customers. Hence, it would be an advancement in the art to provide appealing compositions and / or methods that could resolve, reduce and / or mitigate and / or assist in the resolution, reduction and / or mitigation of inflammation and / or inflammaging and / or the visible effects thereof in a tissue such as the skin.

[0019] Summary of the invention

[0020]

[0017] The inventors have now surprising found that postbiotics derived from Malassezia can advantageously be applied to reduce the concentration of C-C motif chemokine ligand 5 (also abbreviated as “CCL5”) and / or to reduce the concentration of C-C motif chemokine ligand 27 (also abbreviated as “CCL27”) and / or to increase the concentration of Involucrin (also abbreviated as “IVL”) in a tissue such as the human skin and / or the human scalp. Without wishing to be bound by any kind of theory it is believed that the Malassezia postbiotics can thus be advantageous in resolving, reducing and / or mitigating and / or assisting in the resolution, reduction and / or mitigation of inflammation and / or inflammaging and / or the visible effects thereof in a tissue such as the skin.

[0018] Accordingly, in a first aspect, the invention provides a composition comprising one or more Malassezia postbiotics, wherein the Malassezia postbiotics comprise a CCL5 inhibitor and / or a CCL27 inhibitor and / or wherein the Malassezia postbiotics comprise an IVL stimulator. Preferably the composition is a cosmetic and / or non-therapeutic composition.

[0021]

[0019] The composition can advantageously be produced by a process comprising the lysis of the Malassezia microorganisms to yield Malassezia postbiotics and subsequent combination of the Malassezia postbiotics with a cosmetically acceptable carrier. Thus, in a second aspect, the invention also provides a process for the preparation of a cosmetic composition, comprising the steps of:

[0022] - Lysis of one or more Malassezia microorganisms, wherein the one or more Malassezia microorganisms comprise a CCL5 inhibitor and / or a CCL27 inhibitor and / or an IVL stimulator, to yield Malassezia postbiotics; and

[0023] - Combining the Malassezia postbiotics with a cosmetically acceptable carrier.

[0024]

[0020] The composition can advantageously be applied to reduce inflammation and / or inflammaging in a tissue. Hence, in a third aspect, the invention also provides a method of or for the reduction and / or mitigation of inflammation and / or inflammaging and / or the visible effects of inflammation and / or inflammaging, in a tissue, preferably a keratinous tissue such as the skin or scalp, wherein such method comprises the step of application of the above Malassezia postbiotics and / or of the above composition. Preferably the method is a cosmetic and / or non-therapeutic method.

[0025]

[0021] In addition, in a fourth aspect, the invention provides a use of or for the reduction and / or mitigation of inflammation and / or inflammaging and / or the visible effects of inflammation and / or inflammaging, in a tissue, preferably a keratinous tissue such as the skin or scalp, wherein such use comprises the step of application of the above Malassezia postbiotics and / or of the above composition. Preferably the use is a cosmetic and / or non-therapeutic use.

[0026]

[0022] The use of Malassezia postbiotics in the treatment of dandruff is believed to be novel as such. Hence, in a fifth aspect, the invention advantageously also provides a cosmetic composition which is an anti-dandruff composition, wherein such anti-dandruff composition comprising one or more Malassezia postbiotics.

[0027]

[0023] By targeting the molecular mechanisms underlying inflammation and inflammaging, the invention aims to restore skin vitality, improve complexion, reduce the appearance of aging, and / or promote overall skin health.

[0028]

[0024] Advantageously, the above composition, process, method and use thus provide a holistic approach to reduce and / or mitigate and / or assist in the resolution, reduction and / or mitigation of inflammation and / or inflammaging and / or the visible effects thereof in a tissue such as the skin.

[0029]

[0025] In addition, it has surprisingly been found that Malassezia postbiotics derived from Malassezia furfur (also referred to herein as “Malassezia furfur postbiotics”) can reduce lipid and / or lipid-containing sebum production by the skin, whilst Malassezia postbiotics derived from Malassezia restricta (also referred to herein as “Malassezia restricta postbiotics”) and / or derived from Malassezia globosa (also referred to herein as “Malassezia globosa postbiotics”) have an opposite effect and can stimulate lipid and / or lipid-containing sebum production by the skin. Both aspects can be beneficially applied in cosmetic industry. Without wishing to be bound by any kind of theory it is herewith noted that sebum is produced by sebaceous glands, which are part of hair follicles, being part of skin. This sebum comprises or may even consist of lipids.

[0030]

[0026] Therefore, in a sixth aspect, the invention provides a topical composition, preferably a skincare and / or haircare composition and / or a composition to treat, care for or improve the appearance of the skin and / or scalp, more preferably an anti-dandruff composition and / or anti-acne composition, wherein such composition comprises one or more Malassezia postbiotics and wherein the Malassezia postbiotics are derived from Malassezia furfur species. Herein the Malassezia postbiotics, more suitably the Malassezia furfur postbiotics, can suitably be present as an antidandruff agent and / or an anti-acne agent.

[0031]

[0027] Further in a seventh aspect, the invention provides a topical composition, preferably a cosmetic skincare composition for non-therapeutic use, wherein such composition comprises one or more Malassezia postbiotics and wherein the Malassezia postbiotics are derived from Malassezia restricta species, Malassezia globosa species or a mixture thereof; and preferably wherein the composition does not comprise Malassezin. Herein the Malassezia postbiotics, more suitably the Malassezia restricta postbiotics and / or Malassezia globosa postbiotics, can suitably be present as an lipid-stimulating agent and / or barrier-repair agent.

[0032]

[0028] The invention also provides a novel use, wherein such use comprises the step of application of Malassezia postbiotics and / or of the composition as described above wherein the use is a cosmetic and / or non-therapeutic use, preferably

[0033] - a cosmetic use for the treatment of dandruff; and / or

[0034] - a cosmetic use for the treatment of acne; and / or

[0035] - a cosmetic use for stimulating lipid and / or lipid-containing sebum production.

[0036] Brief description of the drawings

[0037]

[0029] The invention is illustrated by the following figures:

[0038] Figure 1 : Schematic representation of Malassezia postbiotics preparation

[0039] Figure 2: Images as obtained in example 3 of the effects of compounds F, R, S and G (respectively Malassezia furfur, M. restricta, M. sympodialis and M. globosa postbiotics) on lipid content.

[0040] Detailed description of the invention

[0041] Definitions

[0042]

[0030] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0043]

[0031] Throughout the present specification and the accompanying claims, the words "comprise" and "include" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0044]

[0032] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, “an element” may mean one element or more than one element. When referring to a noun (e.g. a compound, an additive, etc.) in the singular, the plural is meant to be included. Thus, when referring to a specific moiety, e.g. "gene", this means "at least one" of that gene, e.g. "at least one gene", unless specified otherwise.

[0045]

[0033] When referring to a compound of which several isomers exist (e.g. a D and an L enantiomer), the compound in principle includes all enantiomers, diastereomers and cis / trans isomers of that compound that may be used in the particular aspect of the invention; in particular when referring to such as compound, it includes the natural isomer(s).

[0046]

[0034] Unless explicitly indicated otherwise, the various aspects and embodiments of the invention described herein can be cross-combined.

[0047]

[0035] The term "cosmetic composition " or “cosmetic preparation” refers in the present document to a composition suitable for cosmetic purposes. Suitably a cosmetic composition is herein understood to be a composition for cosmetic, that is, non-therapeutic use. More preferably the term "cosmetic composition " or “cosmetic preparation” refers in the present document to a composition which is applied to the surface of a mammalian keratinous tissue. The terms "cosmetic composition " or “cosmetic preparation” can comprise or consist of those cosmetic compositions as defined underthe heading "Kosmetika" in Rompp Lexikon Chemie, 10th edition 1997, Georg Thieme Verlag Stuttgart, New York as well as to cosmetic compositions as disclosed in A. Domsch, "Cosmetic Compositions", Verlag fur chemische Industrie (ed. H. Ziolkowsky), 4thedition, 1992. More preferably the "cosmetic composition " or “cosmetic preparation” is a cosmetic preparation, respectively a cosmetic composition, that can be topically applied to mammalian keratinous tissue such as e.g. human skin or hair (including eyelashes, the eyebrows, the nails orthe lips), particularly human skin. Hence, the cosmetic composition is preferably a topical composition.

[0048]

[0036] By a topical composition is herein understood a composition for, preferably external, use on keratinous tissue such as the skin.

[0049]

[0037] The term “keratinous tissue" as used in this document refers to tissue containing keratine, more preferably it means the skin (including the body, face, contour of the eyes, scalp), head hair, eyelashes, eyebrows, bodily hairs, nails and / or lips. Preferably, the keratinous tissue is the skin, the scalp, the hair or a combination thereof.

[0050]

[0038] The term “inflammation” as used herein preferably refers to biological responses of a tissue, preferably a keratinous tissue, to injury and / or stress caused by external factors. Such responses include for example redness, swelling, itching, heat and other irritations of the tissue, preferably a keratinous tissue. More preferably the term “inflammation” may refer to a biological response that includes an increased capillary dilation, an increased blood flow and / or an increased vascular permeability in the tissue, and / or a potential leukocytic infiltration of the affected skin. Without wishing to be bound by any kind of theory it is believed that inflammation may be associated with the presence of certain cytokines.

[0051]

[0039] The term “inflammaging”, is used herein to describe a combination of “inflammation” and “aging” and may comprise a chronic, low-grade inflammation of a tissue such as the skin that may accelerate visible signs of aging, such as wrinkles, loss of elasticity, uneven skin tone, and dullness. Without wishing to be bound by any kind of theory inflammaging is believed to be associated with the accumulation of aging skin cells that secrete pro-inflammatory factors, such as certain cytokines.

[0052]

[0040] Cytokines are understood to be small proteins that can for example act as a signalling molecule and can play an important role in the biological response of the body to injury or external stress. Cytokines are believed to be potentially produced by a wide range of cells, including immune cells (like T cells, B cells, macrophages, and dendritic cells), as well as non-immune cells such as endothelial and epithelial cells.

[0053]

[0041] Unless indicated otherwise, preferably “inflamed conditions” are herein understood to refer to cytokine stimulated conditions. More preferably, inflamed conditions are herein understood to refer to conditions wherein the tissue is, and / or was in the preceding 48 hours, in contact with one or more cytokines chosen from the group consisting of lnterleukin-4 (IL-4), Interleukin-13 (IL-13), Interleukin-22 (IL-22), tumor necrosis factor-alpha (TNF-a) and / or any combination thereof. Most preferably inflamed conditions are inflamed conditions as exemplified in the examples.

[0054]

[0042] When referral is made to a certain gene (such as for example the “CCL5 gene”) being "expressed", this may be understood to refer to a biological process of transcription and translation that leads to the production of the corresponding protein (such as for example the corresponding CCL5 protein).

[0055]

[0043] A “subject” as used herein is preferably an individual, more preferably a human individual. The aspects of the invention can be advantageous for all subjects. However, preferably the subject is a female subject. Preferably the subject is a subject having an age of 40 years or more, more preferably an age of 50 years or more.

[0056]

[0044] The terms “active ingredient”, “active agent” or simply “agent” or “active” are used interchangeably herein. These terms preferably referto a substance or ingredient that has a specific physiological or biochemical effect on a keratinous tissue such as the skin, scalp or hair. Such agents may suitably be included in a composition, preferably a cosmetic composition, fortheir ability to target and address specific concerns or provide benefits, such as for example moisturizing, antiaging, brightening, soothing, or protecting. Active agents can thus advantageously be key ingredients responsible for the claimed efficacy of a cosmetic composition.

[0057]

[0045] The terms “helper ingredient”, “helper component” and “excipient” are used interchangeably herein. These terms preferably refer to a substance or ingredient used in a composition to serve as an aid in the processing, stability, and / or delivery of the active ingredients. Excipients can for example be used to ensure the proper form, consistency, and / or stability of a product. Examples of the functions of excipients thus include stabilizing, binding, filling, preserving, facilitating thereof and / or aiding in the delivery of active ingredients. Preferred examples of excipients include stabilizers and / or preservatives.

[0058]

[0046] The terms “adjuvant ingredient”, “adjuvant component” and simply “adjuvant” are used interchangeably herein. These terms preferably refer to a substance or ingredient added to a composition, preferably a cosmetic composition, to enhance the effectiveness of active ingredients. Examples may for example include compounds that improve the efficacy of an active ingredient. An adjuvant may be considered by some to be a subcategory of the excipients.

[0059] The CCL5 inhibitor

[0060]

[0047] The term “CCL5”, as used herein is an abbreviation for chemokine (C-C motif) ligand 5. The CCL5 protein is encoded by the CCL5 gene.

[0061]

[0048] The terms “CCL5 protein” and simply “CCL5” are used interchangeably herein. CCL5 is a protein that can also be referred to as RANTES (Regulated upon Activation, Normal T-cell Expressed and Secreted) protein. CCL5 is proinflammatory chemokine that is believed to be involved in the recruitment of white blood cells to sites of inflammation. Such white blood cells are sometimes also referred to as leukocytes, immune cells or immunocytes. The CCL5 gene is believed to be expressed by T-cells or monocytes. Within the human skin or scalp the CCL5 gene can be expressed by epithelial cells and / or fibroblasts. The CCL5 gene is believed to become upregulated in response to pro-inflammatory signals. After being expressed, CCL5 protein may be secreted into the extracellular space, where it may bind to chemokine receptors (such as CCR1 , CCR3, and CCR5) on target immune cells.

[0062]

[0049] Advantageously the inhibition of the production of CCL5 protein, the CCL5 protein itself and / or one or more of its receptors, such as for example CCR1 , CCR3 and / or CCR5 and preferably CCR5, can help to reduce inflammation and mitigate immune responses.

[0063]

[0050] By a “CCL5 inhibitor” is herein preferably understood a molecule or compound that interferes or can interfere with the production and / or activity of the CCL5 protein. More preferably a “CCL5 inhibitor” is herein understood to be a molecule or compound that inhibits or can inhibit at least partly the production and / or activity of the CCL5 protein. Examples of suitable CCL5 inhibitors may include:

[0064] - molecules or compounds that inhibit or can inhibit, at least partly, the transcription of the CCL5 gene into mRNA or inhibit or can inhibit at least partly the processing, transport, stability or translation of the mRNA;

[0065] - molecules or compounds which degrade or can degrade, at least partly, the CCL5 protein; and

[0066] - molecules or compounds (also referred to as antagonists) which inhibit or can inhibit, at least partly, the action of the CCL5 protein itself, for example by preventing the CCL5 from binding to its receptor(s) (such as for example CCR1 , CCR3 and / or CCR5) or by inhibiting the receptor(s), especially CCR5, in another manner.

[0067] Preferably the inhibition is an inhibition under inflamed conditions. That is, preferably the inhibition is an inhibition under cytokine stimulated conditions. Such may suitably be determined in a manner as exemplified in the examples.

[0068]

[0051] Preferably the CCL5 inhibitor is a molecule or compound that:

[0069] - does not reduce or inhibit the production and / or activity of the CCL5 protein under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a; and

[0070] - does reduce or inhibit the production and / or activity of the CCL5 protein under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0071] Such a reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0072]

[0052] Preferably the CCL5 inhibitor is a molecule or compound of which the presence in or on a tissue, under inflamed conditions, results in a reduction of the concentration of CCL5 within the tissue as compared to the tissue wherein the CCL5 inhibitor is absent.

[0073]

[0053] More preferably the CCL5 inhibitor is a molecule or compound of which the presence in or on a tissue, as compared to the tissue wherein the CCL5 inhibitor is absent,:

[0074] - does not result in a reduction of the concentration of CCL5 within the tissue under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a; and

[0075] - does result in a reduction of the concentration of CCL5 within the tissue under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a. Also here the reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0076]

[0054] Still more preferably the CCL5 inhibitor is a molecule or compound of which the presence in or on a tissue, under inflamed conditions, results in a reduction of the relative expression of the CCL5 gene, as compared to the relative expression of CCL5 gene in the absence of the CCL5 inhibitor, of equal to or more than 40 %, preferably of equal to or more than 50 %, more preferably of equal to or more than 60 %, even more preferably equal to or more than 70 %, still more preferably equal to or more than 75 %, still even more preferably equal to or more than 80%. The relative expression can conveniently be calculated as exemplified in the examples of this disclosure.

[0077]

[0055] Most preferably the CCL5 inhibitor is a molecule or compound of which the presence in or on a tissue, as compared to the tissue, wherein the CCL5 inhibitor is absent,:

[0078] - does not result in a reduction of the relative expression of the CCL5 gene under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a; and

[0079] - results in a reduction of the relative expression of the CCL5 gene of equal to or more than 40 %, preferably of equal to or more than 50 %, more preferably of equal to or more than 60 %, even more preferably equal to or more than 70 %, still more preferably equal to or more than 75 %, still even more preferably equal to or more than 80%, under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0080] Also here the reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0081]

[0056] Without wishing to be bound by any kind of theory it is believed that the inhibition of CCL5 can, in turn, advantageously reduce the activity and / or concentration of white blood cells in the tissue and / or otherwise reduce inflammation and / or mitigate the immune response.

[0082] The CCL27 inhibitor

[0083]

[0057] The term “CCL27”, as used herein is an abbreviation for chemokine (C-C motif) ligand 27. The CCL27 protein is encoded by the CCL27 gene.

[0084]

[0058] The terms “CCL27 protein” and simply “CCL27” are used interchangeably herein. CCL27 is a protein that is also referred to as cutaneous T cell-attracting chemokine, or “CTACK”. CCL27 is a protein that is believed to play a role in the recruitment and therewith activity of T-cells in case of an inflammation of the skin. Within the human skin or scalp, CCL27 proteins are believed to be expressed by keratinocytes, preferably keratinocytes present in the epidermis.

[0085]

[0059] Advantageously the inhibition of CCL27 can help to reduce inflammation and / or inflammaging, such as for example psoriasis and / or atopic dermatitis.

[0086]

[0060] By a “CCL27 inhibitor” is herein preferably understood a molecule or compound that interferes or can interfere with the production and / or activity of the CCL27 protein. More preferably a “CCL27 inhibitor” is herein understood to be a molecule or compound that inhibits or can inhibit at least partly the production and / or activity of the CCL27 protein. Examples of suitable CCL27 inhibitors may include:

[0087] - molecules or compounds that inhibit or can inhibit, at least partly, the transcription of the CCL27 gene into mRNA or inhibit or can inhibit at least partly the processing, transport, stability or translation of the mRNA;

[0088] - molecules or compounds which degrade or can degrade, at least partly, the CCL27 protein; and

[0089] - molecules or compounds (also referred to as antagonists) which inhibit or can inhibit, at least partly, the action of the CCL27 protein itself, for example by preventing the CCL27 from binding to its receptor CCR10 or by inhibiting the receptor CCR10 in another manner.

[0090] Preferably the inhibition is an inhibition under inflamed conditions. That is, preferably the inhibition is an inhibition under cytokine stimulated conditions. Such may suitably be determined in a manner as exemplified in the examples.

[0091]

[0061] Preferably the CCL27 inhibitor is a molecule or compound that:

[0092] - does not reduce or inhibit the production and / or activity of the CCL27 protein, or merely reduces to a limited extent of less than 40 %, under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a; and

[0093] - does reduce or inhibit the production and / or activity of the CCL27 protein, preferably to an extent of equal to or more than 40%, under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0094] Such a reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0095]

[0062] Preferably the CCL27 inhibitor is a molecule or compound of which the presence in or on a tissue, under inflamed conditions, results in a reduction of the concentration of CCL27 within the tissue as compared to the tissue wherein the CCL27 inhibitor is absent.

[0096]

[0063] More preferably the CCL27 inhibitor is a molecule or compound of which the presence in or on a tissue, as compared to the tissue wherein the CCL27 inhibitor is absent,:

[0097] - does not result in a reduction of the concentration of CCL27 within the tissue under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a; and

[0098] - does result in a reduction of the concentration of CCL27 within the tissue under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0099] Also here the reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0100]

[0064] Still more preferably the CCL27 inhibitor is a molecule or compound of which the presence in or on a tissue, under inflamed conditions, results in a reduction of the relative expression of the CCL27 gene, as compared to the relative expression of CCL27 gene in the absence of the CCL27 inhibitor, of equal to or more than 40 %, preferably of equal to or more than 50 %, more preferably of equal to or more than 60 %, even more preferably equal to or more than 70 %, still more preferably equal to or more than 75 %, still even more preferably equal to or more than 80%. The relative expression can conveniently be calculated as exemplified in the examples of this disclosure.

[0101]

[0065] Most preferably the CCL27 inhibitor is a molecule or compound of which the presence in or on a tissue, as compared to the tissue, wherein the CCL27 inhibitor is absent,:

[0102] - does not result in a reduction of the relative expression of the CCL27 gene under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a; and

[0103] - results in a reduction of the relative expression of the CCL27 gene of equal to or more than 40 %, preferably of equal to or more than 50 %, more preferably of equal to or more than 60 %, even more preferably equal to or more than 70 %, still more preferably equal to or more than 75 %, still even more preferably equal to or more than 80%, under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0104] Also here the reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0105]

[0066] Without wishing to be bound by any kind of theory it is believed that the inhibition of CCL27 can, in turn, advantageously reduce inflammation and / or inflammaging.

[0106] The IVL stimulator

[0107]

[0067] The term “IVL”, as used herein is an abbreviation for Involucrin. The IVL protein is encoded by the IVL gene.

[0108]

[0068] The terms “IVL protein” and simply “IVL” are used interchangeably herein. Involucrin is a protein which is important in the formation of cell envelopes to protect so-called corneocyte cells in the skin. Without wishing to be bound by any kind of theory, the IVL protein is believed to be crucial forthe formation of the skin barrier. The cell envelope is believed to provide the mechanical strength to the skin and to help in the protection for example against pollution, pathogens and water loss. Within the human skin or scalp, IVL protein is believed to be expressed by keratinocytes, preferably keratinocytes present in the epidermis, more preferably in the stratum corneum.

[0109]

[0069] Inflammaging is, amongst others, associated with an aging, thinner skin. Without wishing to be bound by any kind of theory it is believed that the IVL stimulator may advantageously stimulate the firmness of the skin and hence may assist in increasing of skin robustness and / or combatting inflammaging.

[0110]

[0070] By an “IVL stimulator” is herein preferably understood a molecule or compound that stimulates, promotes and / or enhances the production and / or activity of the IVL protein. Examples of suitable IVL stimulators may include:

[0111] - molecules or compounds that increase the transcription of the IVL gene into mRNA or increase the processing, transport, stability or translation of the mRNA;

[0112] - molecules or compounds which otherwise increase the production the IVL protein; and

[0113] - molecules or compounds which enhance the stability or activity of the IVL protein.

[0071] Preferably the IVL stimulator is a molecule or compound of which the presence results in an increase of the concentration of IVL within the tissue, preferably a keratinous tissue, as compared to the tissue wherein the IVL stimulator is absent.

[0114]

[0072] More preferably the IVL stimulator is a molecule or compound of which the presence in or on a tissue, as compared to the tissue wherein the IVL stimulator is absent, results in an increase of the concentration of IVL within the tissue under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a. The increase may suitably be determined as exemplified in the examples.

[0115]

[0073] Still more preferably the IVL stimulator is a molecule or compound of which the presence in or on a tissue, preferably under inflamed conditions, results in an increase of the relative expression of the IVL gene, as compared to the relative expression of IVL gene in the absence of the IVL stimulator, of equal to or more than 70 %, preferably of equal to or more than 100 %, more preferably of equal to or more than 150 %, even more preferably equal to or more than 200 %. The relative expression can conveniently be calculated as exemplified in the examples of this disclosure.

[0116]

[0074] Most preferably the IVL stimulator is a molecule or compound of which the presence in or on a tissue, as compared to the tissue wherein the IVL stimulator is absent, results in an increase of the relative expression of the IVL gene of equal to or more than 70 %, preferably of equal to or more than 100 %, more preferably of equal to or more than 150 %, even more preferably equal to or more than 200 %, under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0117] Also here the increase may suitably be determined as exemplified in the examples.

[0118]

[0075] More preferably the IVL stimulator is a molecule or compound of which the presence results in an increase of the relative expression of IVL, as compared to the relative expression of IVL in the absence of the IVL stimulator, of equal to or more than 1 .5 times, preferably of equal to or more than 2 times, more preferably of equal to or more than 3 times, even more preferably equal to or more than 4 times, the relative expression of IVL in the absence of the IVL stimulator. Preferably such increase is an increase under inflamed conditions, more preferably under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a. Also here the relative expression can conveniently be calculated as exemplified in the examples of this disclosure.

[0119] The Malassezia postbiotics

[0120]

[0076] The Malassezia postbiotics in all aspects of the invention are preferably derived from Malassezia species furfur, Malassezia species sympodialis, Malassezia species restricta and / or Malassezia species globosa. That is, preferably Malassezia postbiotics comprise postbiotics derived from or Malassezi

[0121]

[0077] From a cosmetic industry perspective, preferably the Malassezia postbiotics are Malassezia postbiotics derived from Malassezia furfur and / or Malassezia globosa, most preferably derived from Malassezia furfur. Without wishing to be bound by any kind of theory it is believed that Malassezia furfur and / or Malassezia globosa can be produced faster and easier in an industrial setting than Malassezia sympodialis and / or Malassezia restricta. That is, Malassezia furfur and Malassezia globosa have the advantage that they can be more robust and less fragile than Malassezia sympodialis and / or Malassezia restricta , for example in a production at commercial scale. Hence, in a preferred embodiment the Malassezia postbiotics are derived from Malassezia furfur and / or Malassezia globosa, most preferably derived from Malassezia furfur, and produced at a scale of equal to or more than 100 kilograms per year, more preferably equal to or more than 1000 kilograms per year.

[0122]

[0078] From an CCL5 inhibition perspective, preferably the Malassezia postbiotics are Malassezia postbiotics derived from Malassezia furfur, Malassezia sympodialis and I or Malassezia restricta .

[0079] From an CCL27 inhibition perspective, preferably the Malassezia postbiotics are Malassezia postbiotics derived from Malassezia globosa, Malassezia sympodialis and I or Malassezia restricta , most preferably derived from Malassezia restricta.

[0123]

[0080] From an IVL stimulation perspective, preferably the Malassezia postbiotics are Malassezia postbiotics derived from Malassezia furfur, Malassezia sympodialis and I or Malassezia restricta , more preferably derived from Malassezia furfur and I or Malassezia sympodialis, most preferably derived from Malassezia sympodialis.

[0124]

[0081] When taking into account both industry perspective and / or desire for a Malassezia species with a relatively good robustness as well as the desire for a good CCL5 inhibition and / or good CCL27 inhibition and I or sufficient IVL stimulation, the Malassezia postbiotics are most preferably derived from Malassezia globosa.

[0125]

[0082] In one preferred embodiment, the Malassezia postbiotics is a Malassezia postbiotics which comprises a CCL5 inhibitor and a CCL27 inhibitor.

[0126]

[0083] In another preferred embodiment, the Malassezia postbiotics is a Malassezia postbiotics which comprises

[0127] -an IVL stimulator; and

[0128] - a CCL5 inhibitor and / or a CCL27 inhibitor.

[0129]

[0084] Most preferably the Malassezia postbiotics is a Malassezia postbiotics which comprises a CCL5 inhibitor and a CCL27 inhibitor and an IVL stimulator.

[0130]

[0085] Preferences for the CCL5 inhibitor, the CCL27 inhibitor and the IVL stimulator are as described above and below.

[0131]

[0086] In addition to the CCL5 inhibitor and / or a CCL27 inhibitor and / or IVL stimulator the Malassezia postbiotics may comprise several other compounds.

[0132]

[0087] Preferably the Malassezia postbiotics comprise one or more fatty acids, preferably short chain fatty acids such as acetic acid and propionic acid and / or medium chain fatty acids, such as lauric acid, capric acid and caprylic acid. Most preferably the Malassezia postbiotics comprises one or more of lauric acid, capric acid and caprylic acid

[0088] Preferably the Malassezia postbiotics comprise one or more polysaccharides, preferably beta-glucans.

[0133]

[0089] Preferably the Malassezia postbiotics comprise mannan and / or mannosylated glycoproteins.

[0134]

[0090] Preferably the Malassezia postbiotics comprise one or more sphingolipids, more preferably one or more sphingolipids chosen from the group consisting of sphingomyelins, ceramides, phytoceramides, sphingosine-1 -phosphate, glycosphingolipids and combinations thereof.

[0135]

[0091] The Malassezia postbiotics further may or may not comprise Malassezin. Without wishing to be bound to any kind of theory it is believed that Malassezin may induce the production of a pro- inflammatory cytokine such as TNF-a, which may stimulate inflammation. In one preferred embodiment, as Malassezin may stimulate inflammation, the Malassezia postbiotics may preferably not comprise Malassezin. In another preferred embodiment, accepting that some Malassezia postbiotics may comprise Malassezin, the Malassezin-stimulated inflammation may advantageously be offset by the CCL5 inhibitor and / or CCL27 inhibitor and / or IVL stimulator within the Malassezia postbiotics. Without wishing to be bound to any kind of theory it is believed that postbiotics derived from Malassezia furfur may comprise Malassezin. Most preferably the Malassezia postbiotics are therefore not postbiotics derived from Malassezia furfur (also referred to herein as Malassezia furfur postbiotics)

[0136]

[0092] The Malassezia postbiotics further may or may not comprise lndole-3-carboxaldehyde. Most preferably the Malassezia postbiotics comprise lndole-3-carboxaldehyde (I3C).

[0137]

[0093] Preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence results in a reduction of the concentration of CCL5 within a tissue, preferably a keratinous tissue, as compared to the tissue wherein the Malassezia postbiotics is not applied.

[0138]

[0094] Preferably the Malassezia postbiotics is a Malassezia postbiotics that:

[0139] - does not reduce or inhibit the production and / or activity of the CCL5 protein under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a; and

[0140] - does reduce or inhibit the production and / or activity of the CCL5 protein under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0141] Such a reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0142]

[0095] Preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, preferably a keratinous tissue, under inflamed conditions, results in a reduction of the concentration of CCL5 within the tissue as compared to the tissue wherein Malassezia postbiotics is absent.

[0143]

[0096] More preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, preferably a keratinous tissue, as compared to the tissue wherein the Malassezia postbiotics is absent,:

[0144] - does not result in a reduction of the concentration of CCL5 within the tissue under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a; and - does result in a reduction of the concentration of CCL5 within the tissue under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0145] Also here the reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0146]

[0097] Still more preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, preferably a keratinous tissue, preferably under inflamed conditions, results in a reduction of the relative expression of the CCL5 gene of equal to or more than 40 %, preferably of equal to or more than 50 %, more preferably of equal to or more than 60 %, even more preferably equal to or more than 70 %, still more preferably equal to or more than 75 %, still even more preferably equal to or more than 80%, as compared to the relative expression of CCL5 gene in the absence of the Malassezia postbiotics. The relative expression can conveniently be calculated as exemplified in the examples of this disclosure. Preferably the reduction of the relative expression of the CCL5 gene is a reduction of the relative expression under inflamed conditions.

[0147]

[0098] Most preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, preferably a keratinous tissue:

[0148] - under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a, does not result in a reduction of the relative expression of the CCL5 gene as compared to the tissue wherein the Malassezia postbiotics is absent; and

[0149] - under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a, does result in a reduction of the relative expression of the CCL5 gene of equal to or more than 40 %, preferably of equal to or more than 50 %, more preferably of equal to or more than 60 %, even more preferably equal to or more than 70 %, still more preferably equal to or more than 75 %, still even more preferably equal to or more than 80%, as compared to the tissue wherein the Malassezia postbiotics is absent.

[0150] Also here the reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0151]

[0099] Preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence results in a reduction of the concentration of CCL27 within a tissue, preferably a keratinous tissue, as compared to the tissue wherein the Malassezia postbiotics is not applied.

[0152]

[0100] Preferably the Malassezia postbiotics is a Malassezia postbiotics that:

[0153] - does not reduce or inhibit the production and / or activity of the CCL27 protein under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a; and

[0154] - does reduce or inhibit the production and / or activity of the CCL27 protein under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0155] Such a reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0156]

[0101] Preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, preferably a keratinous tissue, under inflamed conditions, results in a reduction of the concentration of CCL27 within the tissue as compared to the tissue wherein Malassezia postbiotics is absent.

[0157]

[0102] More preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, preferably a keratinous tissue, as compared to the tissue wherein the Malassezia postbiotics is absent,:

[0158] - does not result in a reduction of the concentration of CCL27 within the tissue under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a; and

[0159] - does result in a reduction of the concentration of CCL27 within the tissue under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0160] Also here the reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0161]

[0103] Still more preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, preferably a keratinous tissue, preferably under inflamed conditions, results in a reduction of the relative expression of the CCL27 gene of equal to or more than 40 %, preferably of equal to or more than 50 %, more preferably of equal to or more than 60 %, even more preferably equal to or more than 70 %, still more preferably equal to or more than 75 %, still even more preferably equal to or more than 80%, as compared to the relative expression of CCL27 gene in the absence of the Malassezia postbiotics. The relative expression can conveniently be calculated as exemplified in the examples of this disclosure. Preferably the reduction of the relative expression of the CCL27 gene is a reduction of the relative expression under inflamed conditions.

[0162]

[0104] Most preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, preferably a keratinous tissue:

[0163] - under conditions in the absence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a, does not result in a reduction of the relative expression of the CCL27 gene as compared to the tissue wherein the Malassezia postbiotics is absent; and

[0164] - under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a, does result in a reduction of the relative expression of the CCL27 gene of equal to or more than 40 %, preferably of equal to or more than 50 %, more preferably of equal to or more than 60 %, even more preferably equal to or more than 70 %, still more preferably equal to or more than 75 %, still even more preferably equal to or more than 80%, as compared to the tissue wherein the Malassezia postbiotics is absent.

[0165] Also here the reduction or inhibition may suitably be determined as exemplified in the examples. The inhibition may be an at least part inhibition or a full inhibition.

[0166]

[0105] Preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence results in an increase of the concentration of IVL within the tissue, preferably a keratinous tissue, as compared to the tissue wherein the Malassezia postbiotics is absent.

[0167]

[0106] More preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, as compared to the tissue wherein the Malassezia postbiotics is absent, results in an increase of the concentration of IVL within the tissue under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a. The increase may suitably be determined as exemplified in the examples.

[0168]

[0107] Still more preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, preferably under inflamed conditions, results in an increase of the relative expression of the IVL gene, as compared to the relative expression of IVL gene in the absence of the Malassezia postbiotics, of equal to or more than 70 %, preferably of equal to or more than 100 %, more preferably of equal to or more than 150 %, even more preferably equal to or more than 200 %. The relative expression can conveniently be calculated as exemplified in the examples of this disclosure.

[0169]

[0108] Most preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence in or on a tissue, as compared to the tissue wherein the Malassezia postbiotics is absent, results in an increase of the relative expression of the IVL gene of equal to or more than 70 %, preferably of equal to or more than 100 %, more preferably of equal to or more than 150 %, even more preferably equal to or more than 200 %, under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a.

[0170] Also here the increase may suitably be determined as exemplified in the examples.

[0171]

[0109] More preferably the Malassezia postbiotics is a Malassezia postbiotics of which the presence results in an increase of the relative expression of IVL, as compared to the relative expression of IVL in the absence of the Malassezia postbiotics, of equal to or more than 1 .5 times, preferably of equal to or more than 2 times, more preferably of equal to or more than 3 times, even more preferably equal to or more than 4 times, the relative expression of IVL in the absence of the Malassezia postbiotics. Preferably such increase is an increase under inflamed conditions, more preferably under conditions in the presence of a cytokine mixture comprising IL-4, IL-13, IL-22 and TNF-a. Also here the relative expression can conveniently be calculated as exemplified in the examples of this disclosure.

[0172]

[0110] Even more preferably the Malassezia postbiotics is a Malassezia postbiotics of which the application in or on a tissue, preferably a keratinous tissue, preferably under inflamed conditions, results in:

[0173] - a decrease of relative expression of the CCL5 gene and / or a decrease in concentration of the CCL5 protein, as compared to the tissue wherein the Malassezia postbiotics is not applied; and / or

[0174] - a decrease of relative expression of the CCL27 gene and / or a decrease in concentration of the CCL27 protein, as compared to the tissue wherein the Malassezia postbiotics is not applied; and / or

[0175] - an increase of relative expression of the IVL gene and / or an increase in concentration of the IVL protein, as compared to the tissue wherein the Malassezia postbiotics is not applied.

[0176]

[0111] Most preferably the Malassezia postbiotics is a Malassezia postbiotics of which the application in or on a tissue, preferably a keratinous tissue, preferably under inflamed conditions, results in:

[0177] - a decrease of relative expression of the CCL5 gene and / or a decrease in concentration of the CCL5 protein, as compared to the tissue wherein the Malassezia postbiotics is not applied; and - a decrease of relative expression of the CCL27 gene and / or a decrease in concentration of the CCL27 protein, as compared to the tissue wherein the Malassezia postbiotics is not applied; and

[0178] - an increase of relative expression of the IVL gene and / or an increase in concentration of the IVL protein, as compared to the tissue wherein the Malassezia postbiotics is not applied.

[0179] The relative expression can conveniently be calculated as exemplified in the examples of this disclosure. Further preferences are as mentioned above.

[0180] The composition

[0181]

[0112] In a first aspect, the invention provides a composition comprising one or more Malassezia postbiotics, wherein the Malassezia postbiotics comprise a CCL5 inhibitor and / or a CCL27 inhibitor and / or the Malassezia postbiotics comprise an IVL stimulator. Further preferences for the CCL5 inhibitor, the CCL27 inhibitor and the IVL stimulator are as described above and below.

[0182]

[0113] In addition, or in the alternative, the invention advantageously provides a composition comprising one or more Malassezia postbiotics, wherein the one or more Malassezia postbiotics comprise a Malassezia postbiotic which, when applied to a tissue, preferably a keratinous tissue, preferably under inflamed conditions, results in:

[0183] - a decrease of relative expression of the CCL5 gene and / or a decrease in concentration of the CCL5 protein, as compared to the tissue wherein the Malassezia postbiotics is not applied; and / or

[0184] - a decrease of relative expression of the CCL27 gene and / or a decrease in concentration of the CCL27 protein, as compared to the tissue wherein the Malassezia postbiotics is not applied; and / or

[0185] - an increase of relative expression of the IVL gene and / or an increase in concentration of the IVL protein, as compared to the tissue wherein the Malassezia postbiotics is not applied.

[0186] Further preferences for such Malassezia postbiotics are as described above and below.

[0187]

[0114] Preferably the composition is a cosmetic and / or non-therapeutic composition. In a preferred embodiment, the composition is a composition for the reduction and / or mitigation of inflammation and / or inflammaging and / or the visible effects of inflammation and / or inflammaging, in a tissue, preferably a keratinous tissue such as the skin or scalp.

[0188]

[0115] More preferably the composition is a topical composition.

[0189]

[0116] In a preferred embodiment the composition comprises the Malassezia postbiotics in an amount in the range from equal to or more than 0.0025 % w / w to equal to or less than 25 % w / w, more preferably in an amount in the range from equal to or more than 0.05 % w / w to equal to or less than 15 % w / w, even more preferably in an amount in the range from equal to or more than 0.1 % w / w to equal to or less than 10 % w / w, most preferably in an amount in the range from equal to or more than 0.5 % w / w to equal to or less than 5 % w / w, based on the total weight of the composition.

[0190]

[0117] In another, or alternative, embodiment, the composition is a composition for use in the prevention and / or treatment of inflammation and / or an inflammatory disorder of a tissue, preferably a keratinous tissue such as the skin or scalp. Preferably the composition is a composition for use in a therapy in respect of an inflammation and / or inflammatory disorder, preferably an inflammation and / or inflammatory disorder of a tissue, preferably a keratinous tissue, most preferably the skin and / or scalp. The inflammatory disorder may for example be psoriasis and / or seborrheic dermatitis. The use may be in or to a subject in need of such prevention and / or treatment. Preferably the use comprises a topical application, suitably a topical administration, of the composition.

[0191]

[0118] In another, or alternative, embodiment, the composition is a composition for use as a medicament, preferably an anti-inflammatory medicament. The medicament may advantageously be a medicament for use in the prevention and / or treatment of inflammation and / or an inflammatory disorder of a tissue, preferably a keratinous tissue such as the skin or scalp. The inflammatory disorder may for example be psoriasis and / or seborrheic dermatitis. The use may be in a subject in need of such prevention and / or treatment. Preferably such anti-inflammatory medicament is an anti-inflammatory medicament for topical application and preferably the use comprises a topical application of the medicament to the tissue.

[0192]

[0119] In all aspects and embodiments of the invention the composition is preferably a topical composition. Most preferably the composition is a topical composition for application to a keratinous tissue such as the skin or scalp. That is, the compositions according to the invention are preferably applied, more preferably topically applied, to a tissue, preferably a keratinous tissue, more preferably a skin or scalp tissue. More preferably the tissue is a mammal tissue, more preferably a human tissue, most preferably the skin or scalp of a human subject.

[0193]

[0120] Preferences for the Malassezia postbiotics are as described above (for example in the section on Malassezia postbiotics) and below.

[0194]

[0121] Preferably the composition comprises one or more fatty acids, preferably short chain fatty acids such as acetic acid and propionic acid, or an ester and / or salt thereof, and / or medium chain fatty acids, such as lauric acid, capric acid and caprylic acid, or an ester and / or salt thereof. Most preferably the composition, comprises one or more of lauric acid, capric acid and caprylic acid or an ester and / or salt thereof.

[0195]

[0122] Preferably the composition comprises one or more polysaccharides, preferably betaglucans.

[0196]

[0123] Preferably the composition comprises mannan and / or mannosylated glycoproteins

[0197]

[0124] Preferably the composition comprises one or more sphingolipids, more preferably one or more sphingolipids chosen from the group consisting of sphingomyelins, ceramides, phytoceramides, sphingosine-1-phosphate, glycosphingolipids and combinations thereof.

[0198]

[0125] The composition further may or may not comprise Malassezin. Without wishing to be bound to any kind of theory it is believed that Malassezin may induce the production of a pro-inflammatory cytokine such as TNF-a, which may stimulate inflammation. In one preferred embodiment, as Malassezin may stimulate inflammation, the composition may preferably not comprise Malassezin. In another preferred embodiment, accepting that some Malassezia postbiotics may comprise Malassezin, the Malassezin-stimulated inflammation may advantageously be offset by the CCL5 inhibitor and / or CCL27 inhibitor and / or IVL stimulator within the composition. Without wishing to be bound to any kind of theory it is believed that postbiotics derived from Malassezia furfur may comprise Malassezin. Most preferably the composition may therefore not comprise any postbiotics derived from Malassezia furfur (also referred to herein as Malassezia furfur postbiotics)

[0199]

[0126] The composition further may or may not comprise lndole-3-carboxaldehyde. Most preferably the composition comprises lndole-3-carboxaldehyde (I3C). Hence, the invention also provides a composition comprising one or more Malassezia postbiotics, wherein the one or more Malassezia postbiotics comprise lndole-3-carboxaldehyde (I3C). Further preferences are as described above and below.

[0200]

[0127] In addition to the Malassezia postbiotics, the composition may comprise one or more other ingredients. Preferably the composition further comprises niacinamide (vitamin B3).

[0201]

[0128] In addition, the composition can advantageously comprise one or more natural extracts chosen from the group consisting of Aloe Vera extract, Chamomile extract, Green Tea extract, Calendula extract.

[0202]

[0129] Preferably the composition comprises one or more antioxidants, preferably ascorbic acid and / or tocopherol. Advantageously the composition may comprise a mixture of tocopherol, argania spinosa kernel oil, spent grain wax and / or butyrospermum parkii butter extract (also known as shea butter). Such mixture is commercially available from Dsm-Firmenich as STIMU-TEX® -AS.

[0203]

[0130] Preferably the composition comprises vitamin B12 (also known as cyanobalamin)

[0204]

[0131] Preferably the composition further may further comprise zinc pyrithione, piroctone olamine, salicylic acid, coal tar, ketoconazole, Climbazole, selenium sulfide and / or Ciclopirox Olamine. Without wishing to be bound by any kind of theory it is believed that such additional ingredients may for example be advantageous for treating conditions such as dandruff.

[0205]

[0132] As indicated above, the composition is preferably a cosmetic composition. The term ‘cosmetic composition’ as used herein refers to compositions, which are used to treat, care for or improve the appearance of the skin and / or the scalp. Particular advantageous cosmetic compositions according to the present invention are skincare and / or haircare compositions.

[0206]

[0133] An especially preferred composition according to this invention is a cosmetic composition for the treatment of dandruff. Hence, preferably the composition is a cosmetic anti-dandruff composition. The use of Malassezia postbiotics in the treatment of dandruff is believed to be novel as such.

[0207] Hence, in a further aspect the invention advantageously also provides a composition which is an anti-dandruff composition, wherein such anti-dandruff composition comprises one or more Malassezia postbiotics.

[0208] The one or more Malassezia postbiotics are preferably derived from Malassezia species furfur, Malassezia species sympodialis, Malassezia species restricta and / or Malassezia species globosa. Further preferences for the Malassezia postbiotics and / or the composition are as described above and below.

[0209]

[0134] Preferably such cosmetic compositions comprise a cosmetically acceptable carrier. The term ‘cosmetically acceptable carrier’ (also referred to herein as simply “carrier”) refers to all vehicles / carriers conventionally used in cosmetic compositions, i.e. which are suitable for topical application to the keratinous tissue, have good aesthetic properties, are compatible with the actives present in the composition, and will not cause any unreasonable safety or toxicity concerns. Such carriers are well-known to one of ordinary skill in the art. Examples of carriers include water and oil. Preferably the cosmetically acceptable carrier is water, oil, or a mixture of water and oil.

[0210]

[0135] Preferably the composition is a cosmetic composition, and such cosmetic composition comprises from about 50% to about 99%, preferably from about 60% to about 98%, more preferably from about 70% to about 98%, such as in particular from about 80% to about 95% of a carrier, based on the total weight of the cosmetic composition. In a particular advantageous embodiment, the carrier consists furthermore of at least 40 wt.-%, more preferably of at least 50 wt.-%, most preferably of at least 55 wt.-% of water, such as in particular of about 55 to about 90 wt.-% of water.

[0211]

[0136] The cosmetic composition can advantageously be in the form of a liquid, lotion, cream, foam, scrub, gel, shampoo, conditioner, soap bar or toner, or applied with an implement or via a face or hair mask, pad or patch. Non-limiting examples of such compositions include leave-on and / or rinse-off skin lotions and creams, shampoos, conditioners, shower gels, face wash’s, body wash’s, toilet bars, antiperspirants, deodorants, depilatories, lipsticks, foundations, mascara, sunless tanners and sunscreen lotions. Preferred cosmetic compositions according to the invention are haircare compositions.

[0212]

[0137] Especially preferred are cosmetic compositions that are used in the treatment of dandruff. Hence, in an especially preferred embodiment the cosmetic composition is an anti-dandruff lotion, an anti-dandruff cream or an anti-dandruff shampoo. The invention thus advantageously also provides an anti-dandruff lotion, an anti-dandruff cream or an anti-dandruff shampoo, which comprises one or more Malassezia postbiotics. The one or more Malassezia postbiotics are preferably derived from Malassezia species furfur, Malassezia species sympodialis, Malassezia species restricta and / or Malassezia species globosa. Further preferences for the Malassezia postbiotics and / or the composition are as described above and below.

[0213]

[0138] The cosmetic compositions (including the carrier) according to the invention may comprise conventional adjuvants and additives, such as preservatives / antioxidants, fatty substances / oils, organic solvents, silicones, thickeners, softeners, emulsifiers, antifoaming agents, aesthetic components such as fragrances, surfactants, fillers, anionic, cationic, nonionic or amphoteric polymers or mixtures thereof, propellants, acidifying or basifying agents, dyes, colorings / colorants, abrasives, absorbents, chelating agents and / or sequestering agents, essential oils, skin sensates, astringents, pigments or any other ingredients usually formulated into such compositions.

[0214]

[0139] In accordance with the present invention, such cosmetic compositions may also comprise further cosmetically active ingredients conventionally used in cosmetic compositions. Exemplary active ingredients encompass skin lightening agents; UV-filters, agents for the treatment of hyperpigmentation; other agents (i.e. other than the Malassezia postbiotics) for the prevention or reduction of inflammation; firming, moisturizing, soothing, and / or energizing agents as well as agents to improve elasticity and skin barrier.

[0140] Preferably the composition comprises one or more human milk oligosaccharides as an additional ingredient.

[0215]

[0141] The combination of Malassezia postbiotics and human milk oligosaccharides (HMO) is novel on its own. The invention therefore further provides a composition comprising:

[0216] - one or more Malassezia postbiotics, preferably one or more Malassezia postbiotics derived from Malassezia species furfur, Malassezia species sympodialis, Malassezia species restricta, Malassezia species globosa or a mixture thereof; and

[0217] - one or more human milk oligosaccharides.

[0218] Preferably such a composition is a cosmetic composition. Preferably the composition is a topical composition. Further preferences for the Malassezia postbiotics and the composition are as described herein above and below. Preferably the one or more human milk oligosaccharides (HMO) are chosen from the group consisting of 2'-fucosyllactose (2'FL), lacto-N-neotetraose (LNnT), 3- fucosyllactose (3FL), difucosyllactose (DFL), Lacto-N-fucopentaose I (LNFP I), 3'Sialyllactose Sodium Salt (3'SL), 6'Sialyllactose Sodium Salt (6'SL), Lacto-N-Tetraose (LNT) and combinations thereof. Most preferably the one or more human milk oligosaccharides (HMO) are Lacto-N- neotetraose (LNnT) or 3-Sialyllactose (3’SL) or a combination thereof.

[0219] In such a composition the weight ratio between the one or more human milk oligosaccharides and the Malassezia postbiotics may vary widely. Preferably the weight ratio of the total weight of human milk oligosaccharides to the total weight of Malassezia postbiotics varies in the range between 1 :100 to 100:1 , more preferably between 1 :10 to 100:1 , most preferably between 1 :5 to 50:1 .

[0220]

[0142] As explained by Pagac et al, in their abstract titled “Topical application of human milk oligosaccharides alleviates chronic inflammation of postmenopausal skin”, hormonal changes during menopause can drastically affect the incidence and severity of rosacea, a chronic inflammatory skin condition that causes long-term redness on the face and leads to psychological distress. Pagac et al. state that while prescription medicines help to reduce symptoms, an effective cure for rosacea does not exist and they identify an unmet need to manage such skin disorders associated with menopausal status. Subsequently Pagac et al. elaborate that in a placebo-controlled and split-face clinical trial two biotechnologically produced HMOs, Lacto-N-neotetraose (LNnT) and 3-Sialyllactose (3’SL) were investigated for reduction of rosacea- associated symptoms. (The study cohort consisted of Caucasian (n=63), postmenopausal female volunteers (aged 52-60), diagnosed with mild rosacea (subtype l / ll)). As a result it was found that topical application of either of the two HMO structures significantly reduced inter alia the variations in facial skin redness (after 56 days, 5.1 %, p<0.05, compared to placebo), TEWL (18.6%, p<0.001), sebum (45.9%, p<0.01), skin surface pH (after 7 days, 5.8%, p<0.05) and temperature (1 .2%, p<0.001), increased skin hydration (after 28 days, 12.9%, p<0.05) and microcirculation (after 56 days, 6%, p<0.05), and improved clinician’s erythema assessment (CEA) scores (after 7 days, 13.1 %, p<0.05).

[0221] Pagac et al. concluded that human milk oligosaccharides (HMO) possessed beneficial effects when applied as a novel class of topical skin care active ingredients tailored for postmenopausal women suffering from rosacea-induced inflammatory symptoms. The abstract of Pagac et al. is included herein by reference.

[0222]

[0143] Further the cosmetic composition may comprise cosmetic excipients, diluents, adjuvants, additives. Examples of cosmetic excipients, diluents, adjuvants, additives as well as active ingredients commonly used in the skin care industry which are suitable for use in the cosmetic compositions of the present invention are for example described in the International Cosmetic Ingredient Dictionary & Handbook by Personal Care Product Council (http: / / www.personalcarecouncil.org / ), accessible by the online INFO BASE (http: / / online.personalcarecouncil.org / jsp / Home.jsp), without being limited thereto.

[0223]

[0144] Preferred further cosmetic ingredients include for example panthenol and / or hydroxyacetophenone.

[0224] The process

[0225]

[0145] The invention also provides a process for the preparation of a cosmetic composition, comprising the steps of:

[0226] - Lysis of one or more Malassezia microorganisms, wherein the one or more Malassezia microorganisms comprise a CCL5 inhibitor and / or a CCL27 inhibitor and / or an IVL stimulator, to yield Malassezia postbiotics; and

[0227] - Combining the Malassezia postbiotics with a cosmetically acceptable carrier.

[0228]

[0146] More preferably the process is a process for the preparation of a cosmetic composition, comprising the steps of: a) Growing a Malassezia culture, preferably a Malassezia restricta culture, Malassezia globosa culture, Malassezia sympodialis culture, a Malassezia furfur culture or a culture comprising a mixture thereof, to yield a Malassezia culture; b) Harvesting cells of the Malassezia culture, preferably in pellet form, to yield Malassezia cells; c) Lysing the Malassezia cells, preferably with the help of a lysis system and / or grinding, to yield cell lysates. d) Collecting the Malassezia postbiotics from the cell lysates, preferably by centrifuging, or otherwise separating, the cell lysates into a supernatant and a residue; e) Combining the Malassezia postbiotics with a cosmetically acceptable carrier and optionally one or more adjuvants and / or additives to yield a cosmetic composition.

[0229] Advantageously the invention also provides a cosmetic composition obtained or obtainable by such a process.

[0230]

[0147] Preferences for the Malassezia postbiotics, the steps and the cosmetically acceptable carrier and optionally one or more adjuvants and / or additives are as described above and below, amongst others in the examples.

[0231] The method

[0148] The above Malassezia postbiotics and / or of the above composition can be advantageous for reducing the concentration of CCL5 and / or CCL27 and / or for increasing the concentration of IVL in a tissue such as the human skin and / or scalp.

[0232]

[0149] In a further aspect, the invention thus provides a method of decreasing the expression of the CCL5 gene and / or the concentration of the CCL5 protein; and / or decreasing the expression of the CCL27 gene and / or the concentration of the CCL27 protein; and / or of increasing the expression of the IVL gene and / or the concentration of the IVL protein, in a tissue, preferably a keratinous tissue, wherein such method comprises the step of application of the above Malassezia postbiotics and / or of the above composition. The application is preferably a topical application.

[0233]

[0150] Preferably the method is a cosmetic and / or non-therapeutic method. The application is preferably a topical application.

[0234]

[0151] In one embodiment, the invention provides a method of or for the reduction and / or mitigation of inflammation and / or inflammaging and / or the visible effects of inflammation and / or inflammaging, in a tissue, preferably a keratinous tissue such as the skin or scalp, wherein such method comprises the step of application of the above Malassezia postbiotics and / or of the above composition. Preferably the method is a cosmetic and / or non-therapeutic method. Preferably the method comprises a topical application of the Malassezia postbiotics and / or the composition.

[0235]

[0152] An especially preferred method according to this invention is a cosmetic method for the treatment of dandruff, wherein such method comprises the step of application of one or more Malassezia postbiotics. Preferably such a cosmetic method for the treatment of dandruff comprises the step of application of one or more compositions as described herein. The one or more Malassezia postbiotics are preferably derived from Malassezia species furfur, Malassezia species sympodialis, Malassezia species restricta and / or Malassezia species globosa. Further preferences for the Malassezia postbiotics and / or such compositions in such a treatment are as described above and below.

[0236]

[0153] In another, or alternative, embodiment, the method is a method of or for the prevention and / or treatment of inflammation and / or an inflammatory disorder of a tissue, preferably a keratinous tissue such as the skin or scalp, wherein such method comprises the step of application of the above Malassezia postbiotics and / or of the above composition. The inflammatory disorder may for example be psoriasis and / or seborrheic dermatitis. The method is preferably a method of or for the prevention and / or treatment of inflammation and / or an inflammatory disorder of a tissue in a subject in need of such prevention and / or treatment. That is, the method may suitably be a method wherein the Malassezia postbiotics and / or of the above composition is / are applied, preferably topically administered, in or to a subject in need of such prevention and / or treatment. Preferably the method comprises a topical application of the Malassezia postbiotics and / or the composition.

[0237]

[0154] Preferences for Malassezia postbiotics and for the composition are as described above and below. The use

[0238]

[0155] In addition, the invention provides a use in or for the purpose of decreasing the expression of the CCL5 gene and / or the concentration of the CCL5 protein; and / or decreasing the expression of the CCL27 gene and / or the concentration of the CCL27 protein; and / or of increasing the expression of the IVL gene and / or the concentration of the IVL protein, in or on a tissue, preferably a keratinous tissue, wherein such use is a use of the above Malassezia postbiotics and / or of the above composition in or on a tissue, preferably a keratinous tissue.

[0239]

[0156] Preferably the use is a topical use.

[0240]

[0157] Preferably the use is a cosmetic and / or non-therapeutic use. In one embodiment, the invention provides a use of or for the reduction and / or mitigation of inflammation and / or inflammaging and / or the visible effects of inflammation and / or inflammaging, in a tissue, preferably a keratinous tissue such as the skin or scalp, wherein such use comprises the step of application of the above Malassezia postbiotics and / or of the above composition. Preferably such use comprises a topical application of the Malassezia postbiotics and / or the composition.

[0241]

[0158] An especially preferred use according to this invention is a cosmetic use for the treatment of dandruff. The use of Malassezia postbiotics in or for the treatment of dandruff is believed to be novel as such. Hence the invention also provides a novel cosmetic use in or for the treatment of dandruff, wherein such use comprises the step of application of one or more Malassezia postbiotics. Preferably such a cosmetic use for the treatment of dandruff comprises the step of application of one or more compositions as described herein. The one or more Malassezia postbiotics are preferably derived from Malassezia species furfur, Malassezia species sympodialis, Malassezia species restricta and / or Malassezia species globosa. Further preferences for the Malassezia postbiotics and / or such compositions in such a treatment are as described above and below.

[0242]

[0159] In another, or alternative, embodiment, the use is a use of or for the prevention and / or treatment of inflammation and / or an inflammatory disorder of a tissue, preferably a keratinous tissue such as the skin or scalp, wherein such use comprises the step of application of the above Malassezia postbiotics and / or of the above composition. The inflammatory disorder may for example be psoriasis and / or seborrheic dermatitis. The use is preferably a use of or for the prevention and / or treatment of inflammation and / or an inflammatory disorder of a tissue in a subject in need of such prevention and / or treatment. That is, the use may suitably be a use wherein the Malassezia postbiotics and / or of the above composition is / are applied, preferably topically administered, in or to a subject in need of such prevention and / or treatment.

[0243]

[0160] Most preferably the use comprises a topical application of the Malassezia postbiotics and / or the composition.

[0244]

[0161] Preferences for Malassezia postbiotics and for the composition are as described above and below.

[0245] The use of Malassezia furfur postbiotics as anti-dandruff and / or anti-acne agent

[0162] Dandruff is an undesired skin condition of the scalp that often results in an inflamed and itchy skin and the presence of white flakes scattered throughout the hair. This condition affects about 50% of the adult population. While dandruff is neither contagious nor medically serious, its appearance is undesirable and can cause distress and embarrassment for those affected.

[0246]

[0163] Without wishing to be bound by any kind of theory it is believed that dandruff is caused by a complex system of biological factors, wherein a fungus feeds on scalp oils, breaking them down and producing oleic acid. Many individuals are sensitive to oleic acid, prompting the body to accelerate the renewal of skin cells in an attempt to eliminate the irritant. This accelerated renewal of skin cells is believed to be the cause of the formation of the flakes, which are dead skin cells that accumulate visibly on the scalp or shoulders. Still there is no full understanding of the systems involved yet.

[0247]

[0164] Initially it was believed that the most significant cause of dandruff was the overgrowth of the fungal species Malassezia furfur. However, more recently studies have identified Malassezia restricta as the key cause. For example, Rudramurthy et al, in their article titled “Association of Malassezia species with dandruff', published in Indian J Med Res 139, March 2014, pages 431- 437 concluded that the predominant species causing dandruff in Indian population appeared to be M. globosa and M. restricta as they found that a high density of these species on the scalp was related with the severity of dandruff. Pagac et al, in their article titled “A New Generation of Postbiotics for Skin and Scalp: In Situ Production of Lipid Metabolites by Malassezia", published in Microorganisms 2024, vol.12, pages 1-17, refer to a recent study indeed showing that Malassezia restricta mediates the production of sebum peroxidation products acting on the epidermis and altering skin barrier functions, thereby potentially triggering and maintaining dandruff.

[0248]

[0165] The most well known treatment for dandruff has been the use of pyrithione, often applied as zinc pyrithione (ZPT).

[0249]

[0166] However, the desirability of pyrithione in anti-dandruff shampoos has recently declined sharply. Customers tend to avoid pyrithione as it is perceived as less safe. In certain jurisdictions the zinc pyrithione has even been banned due to concerns about potential health risks and environmental toxicity. Also, some consumers are experiencing irritation, dryness, or allergic reactions when using products containing pyrithione, leading to a call for gentler alternatives.

[0250]

[0167] In addition, the public has raised sustainability issues in respect of the pyrithione and producers of anti-dandruff haircare products are therefore looking for more eco-friendly “natural” or “nature-identical” alternatives.

[0251]

[0168] There is therefore a need for alternative personal care agents, such as alternative haircare agents, for example alternative anti-dandruff agents, suitable for use in cosmetic compositions such as an anti-dandruff shampoo.

[0252]

[0169] As described above, the invention also advantageously provides a use of Malassezia postbiotics derived from Malassezia furfur species (also referred to herein as “Malassezia furfur postbiotics”) as an anti-dandruff agent. That is, the invention provides Malassezia furfur postbiotics, more preferably a composition comprising Malassezia furfur postbiotics, for use in a method of treating dandruff. Suitably the invention therefore also provides the use of Malassezia furfur postbiotics in the manufacture of a composition for the treatment of dandruff. In addition, the invention provides the use of a composition comprising Malassezia furfur postbiotics for the treatment of dandruff.

[0253]

[0170] As explained above, for many such dandruff is not medically serious, but merely its appearance is undesirable and a cosmetic treatment rather than a medical treatment is desired.

[0254]

[0171] The invention also provides the simple use of Malassezia postbiotics derived from Malassezia furfur species (also referred to herein as “Malassezia furfur postbiotics”), more preferably the use of a composition comprising Malassezia furfur postbiotic, for the reduction of lipid production and / or lipid-containing sebum production, for example by the skin and / or scalp. Alternatively phrased, the invention also provides Malassezia furfur postbiotics, more preferably a composition comprising Malassezia furfur postbiotics, for use as a lipid production inhibitor and / or sebum production inhibitor and / or for use in a method to reduce and / or inhibit lipid production and / or sebum production, for example by the skin and / or scalp.

[0255]

[0172] Therefore, in a further aspect, the invention provides a topical composition, preferably a haircare composition and / or a composition to treat, care for or improve the appearance of the scalp, more preferably an anti-dandruff composition, wherein such composition comprises one or more Malassezia postbiotics and wherein the Malassezia postbiotics are derived from Malassezia furfur species. Herein the Malassezia postbiotics, more suitably the Malassezia furfur postbiotics, can suitably be present as an anti-dandruff agent.

[0256]

[0173] Preferences for such a composition are as described elsewhere herein. For example, the composition may or may not comprise Malassezin.

[0257]

[0174] In a preferred embodiment the topical composition comprises the Malassezia furfur postbiotics in an amount in the range from equal to or more than 0.0025 % w / w to equal to or less than 25 % w / w, more preferably in an amount in the range from equal to or more than 0.05 % w / w to equal to or less than 15 % w / w, even more preferably in an amount in the range from equal to or more than 0.1 % w / w to equal to or less than 10 % w / w, most preferably in an amount in the range from equal to or more than 0.5 % w / w to equal to or less than 5 % w / w, based on the total weight of the composition.

[0258]

[0175] Such topical composition, more preferably a topical anti-dandruff composition, preferably further comprises one or more of the group consisting of pyrithione salts, piroctone olamine, tea tree oil, ketoconazole, selenium sulfide, climbazole, hexamidine diisethionate, dichlorobenzyl alcohol (DCBA), coal tar, azelaic acid, salicylic acid, undecylenic acid, 18-B-Glycyrrhetinic Acid and mixtures thereof, more preferably one or more of the group consisting of piroctone olamine, tea tree oil, ketoconazole, selenium sulfide, climbazole, hexamidine diisethionate, dichlorobenzyl alcohol (DCBA), coal tar, azelaic acid, salicylic acid, undecylenic acid, 18-B-Glycyrrhetinic Acid and mixtures thereof.

[0259]

[0176] Further the topical composition may comprise cosmetic excipients, diluents, adjuvants, additives. Examples of cosmetic excipients, diluents, adjuvants, additives as well as active ingredients commonly used in the skin care industry which are suitable for use in the cosmetic compositions of the present invention are for example described in the International Cosmetic Ingredient Dictionary & Handbook by Personal Care Product Council (http: / / www.personalcarecouncil.org / ), accessible by the online INFO BASE (http: / / online.personalcarecouncil.org / jsp / Home.jsp), without being limited thereto.

[0260]

[0177] Preferably the topical composition includes for example panthenol and / or hydroxyacetophenone.

[0261]

[0178] In a preferred embodiment, the topical composition may in addition comprise other actives and / or excipients, such as for example compounds chosen from the group consisting of niacinamide; glycerin; pentavitin; alkanediols; such as propylene glycol, 1 ,2-pentylene glycol, 1 ,2- hexandiol, 1 ,2-heptandiol, 1 ,2-octandiol, caprylyl glycol, glyceryl caprylate, propane-1 ,3-diol and / or propane-1 ,2-diol; fragrances; and / or carriers, such as water and / or glycerol. Further preferences are as exemplified elsewhere in this specification.

[0262]

[0179] The topical composition can advantageously be in the form of a liquid, lotion, cream, foam, scrub, gel, shampoo, conditioner, soap bar or toner. Non-limiting examples of such compositions include leave-on and / or rinse-off skin lotions and creams, shampoos, conditioners, shower gels. Preferred topical compositions include haircare compositions, most preferably anti-dandruff shampoos.

[0263]

[0180] Acne is also an undesired skin condition, wherein sebum and lipid metabolism can play a central role. Without wishing to be bound by any kind of theory, it is believed that in acne-prone skin, sebaceous glands produce increased amounts of sebum with an altered lipid composition, promoting follicular blockage and creating an environment favorable for undesired micro-organisms such as specific pathogenic Cutibacterium acnes. Similar to dandruff, while acne is neither contagious nor medically serious, its appearance is undesirable and can cause distress and embarrassment forthose affected. There thus exists a continuous desire for novel anti-acne agents, for example for use in cosmetic compositions such as an facial cremes.

[0264]

[0181] As described above, the invention also advantageously provides a use of Malassezia postbiotics derived from Malassezia furfur species (also referred to herein as “Malassezia furfur postbiotics”) as an anti-acne agent. That is, the invention provides Malassezia furfur postbiotics, more preferably a composition comprising Malassezia furfur postbiotics, for use in a method of treating acne. Suitably the invention therefore also provides the use of Malassezia furfur postbiotics in the manufacture of a composition for the treatment of acne. In addition, the invention provides the use of a composition comprising Malassezia furfur postbiotics for the treatment of acne.

[0265]

[0182] As explained above, for many such acne is not medically serious, but merely its appearance is undesirable and a cosmetic treatment rather than a medical treatment is desired.

[0266]

[0183] The invention also provides the simple use of Malassezia postbiotics derived from Malassezia furfur species (also referred to herein as “Malassezia furfur postbiotics”), more preferably the use of a composition comprising Malassezia furfur postbiotic, for the reduction of lipid production and / or lipid-containing sebum production, for example by facial skin. Alternatively phrased, the invention also provides Malassezia furfur postbiotics, more preferably a composition comprising Malassezia furfur postbiotics, for use as a lipid production inhibitor and / or sebum production inhibitor and / or for use in a method to reduce and / or inhibit lipid production and / or sebum production, for example by facial skin.

[0267]

[0184] Therefore, in a further aspect, the invention provides a topical composition, preferably a skincare composition and / or a composition to treat, care for or improve the appearance of (parts of) the facial skin, more preferably an anti-acne composition, wherein such composition comprises one or more Malassezia postbiotics and wherein the Malassezia postbiotics are derived from Malassezia furfur species. Herein the Malassezia postbiotics, more suitably the Malassezia furfur postbiotics, can suitably be present as an anti-acne agent.

[0268]

[0185] Preferences for such a composition are as described elsewhere herein. For example, the composition may or may not comprise Malassezin.

[0269]

[0186] In a preferred embodiment the topical composition comprises the Malassezia furfur postbiotics in an amount in the range from equal to or more than 0.0025 % w / w to equal to or less than 25 % w / w, more preferably in an amount in the range from equal to or more than 0.05 % w / w to equal to or less than 15 % w / w, even more preferably in an amount in the range from equal to or more than 0.1 % w / w to equal to or less than 10 % w / w, most preferably in an amount in the range from equal to or more than 0.5 % w / w to equal to or less than 5 % w / w, based on the total weight of the composition.

[0270]

[0187] Such topical composition, more preferably a topical anti-acne composition, preferably further comprises other topical anti-acne agents, for example one or more compounds selected from the group consisting of retinoids (such as tretinoin, adapalene, and tazarotene), benzoyl peroxide, salicylic acid, clindamycin, erythromycin, niacinamide, azelaic acid, tea tree oil, glycolic acid, lactic acid, and dapsone and mixtures thereof.

[0271]

[0188] Further the topical composition may comprise cosmetic excipients, diluents, adjuvants, additives. Examples of cosmetic excipients, diluents, adjuvants, additives as well as active ingredients commonly used in the skin care industry which are suitable for use in the cosmetic compositions of the present invention are for example described in the International Cosmetic Ingredient Dictionary & Handbook by Personal Care Product Council (http: / / www.personalcarecouncil.org / ), accessible by the online INFO BASE (http: / / online.personalcarecouncil.org / jsp / Home.jsp), without being limited thereto.

[0272]

[0189] Preferably the topical composition includes for example panthenol and / or hydroxyacetophenone.

[0273]

[0190] In a preferred embodiment, the topical composition may in addition comprise other actives and / or excipients, such as for example compounds chosen from the group consisting of niacinamide; glycerin; pentavitin; alkanediols; such as propylene glycol, 1 ,2-pentylene glycol, 1 ,2- hexandiol, 1 ,2-heptandiol, 1 ,2-octandiol, caprylyl glycol, glyceryl caprylate, propane-1 ,3-diol and / or propane-1 ,2-diol; fragrances; and / or carriers, such as water and / or glycerol. Further preferences are as exemplified elsewhere in this specification.

[0274]

[0191] The topical composition can advantageously be in the form of a liquid, lotion, cream, foam, scrub, gel, shampoo, conditioner, soap bar or toner, or applied with an implement or via a face mask, pad or patch. Non-limiting examples of such compositions include leave-on and / or rinse-off skin lotions and creams, shampoos, conditioners, shower gels, face wash’s, body wash’s, and soap bars. Preferred topical compositions according to the invention are skincare compositions.

[0275] The use of Malassezia restricta postbiotics and / or Malassezia qlobasa postbiotics for treating dry skin

[0276]

[0192] Lipids can play an important role in maintaining the skin's natural barrier, helping to lock in moisture and prevent dehydration. From a cosmetic perspective, higher lipid levels can contribute to a more hydrated and smooth appearance of the skin. This is especially beneficial for individuals with dry or sensitive skin, such as many postmenopausal individuals. Increased lipid production by the skin can provide added protection against environmental stressors and can help to maintain skin’s elasticity. Moreover, an increased production of lipids and / or lipid-containing sebum can give the skin a healthier, more radiant glow, reducing the appearance of fine lines and promoting a more youthful complexion. In addition, lipids can support the skin’s ability to repair itself, leading to improved texture and a stronger skin barrier.

[0277]

[0193] As explained above inventors have surprisingly found that Malassezia postbiotics derived from Malassezia restricta (also referred to herein as “Malassezia restricta postbiotics”) and / or derived from Malassezia globosa (also referred to herein as “Malassezia globosa postbiotics”) can stimulate lipid and / or sebum production by the skin. Such can be beneficially applied in cosmetic industry.

[0278]

[0194] As described above, the invention also advantageously provides a use of Malassezia postbiotics derived from Malassezia restricta (also referred to herein as “Malassezia restricta postbiotics”) and / or derived from Malassezia globosa (also referred to herein as “Malassezia globosa postbiotics”) as a lipid-stimulating agent and / or barrier-repair agent. That is, the invention provides Malassezia restricta postbiotics and / or Malassezia globosa postbiotics, more preferably a composition comprising Malassezia restricta postbiotics and / or Malassezia globosa postbiotics, for use in a method of treating lipid-depleted skin and / or dry skin. Suitably the invention therefore also provides the use of Malassezia restricta postbiotics and / or Malassezia globosa postbiotics in the manufacture of a composition for the treatment of lipid-depleted and / or dry skin. In addition, the invention provides the use of a composition comprising Malassezia restricta postbiotics and / or Malassezia globosa postbiotics for the treatment of lipid-depleted and / or dry skin.

[0279]

[0195] As explained above, for many such a lipid-depleted or dry skin is not medically serious, but merely its appearance is undesirable and a cosmetic treatment rather than a medical treatment is desired.

[0196] The invention also provides the simple use of Malassezia postbiotics derived from Malassezia restricta (also referred to herein as “Malassezia restricta postbiotics”) and / or derived from Malassezia globosa (also referred to herein as “Malassezia globosa postbiotics”), more preferably the use of a composition comprising Malassezia restricta postbiotics and / or Malassezia globosa postbiotics, for the stimulation, promotion and / or increase of lipid production and / or lipid- containing sebum production, for example by the skin. Alternatively phrased, the invention also provides Malassezia restricta postbiotics and / or Malassezia globosa postbiotics, more preferably a composition comprising Malassezia restricta postbiotics and / or Malassezia globosa postbiotics, for use as a lipid production stimulator and / or lipid production promoter and / or for use in a method to stimulate and / or promote and / or increase lipid production, for example by the skin. Such can for example be very advantageous for the facial skin or / or body skin, such as the skin of hands and / or legs..

[0280]

[0197] In a further aspect, the invention therefore provides a topical composition, preferably a cosmetic skincare composition for non-therapeutic use, wherein such composition comprises one or more Malassezia postbiotics and wherein the Malassezia postbiotics are derived from Malassezia restricta species, Malassezia globosa species or a mixture thereof; and preferably wherein the composition does not comprise Malassezin. Herein the Malassezia postbiotics, more suitably the Malassezia restricta postbiotics and / or Malassezia globosa postbiotics, can suitably be present as an lipid-stimulating agent and / or barrier-repair agent.

[0281]

[0198] Preferences for such a composition are as described elsewhere herein.

[0282]

[0199] Such topical composition may or may not comprise Malassezin. More preferably such topical composition comprising Malassezia restricta postbiotics and / or Malassezia globosa postbiotics does not comprise Malassezin. That is, preferably and advantageously such topical composition comprising Malassezia restricta postbiotics and / or Malassezia globosa postbiotics is free of Malassezin. Without wishing to be bound to any kind of theory, it is believed that such Malassezin may have negative effects on an already dry and / or lipid-depleted skin.

[0283]

[0200] In a preferred embodiment the topical composition comprises the Malassezia restricta postbiotics and / or Malassezia globosa in a total amount of in the range from equal to or more than 0.0025 % w / w to equal to or less than 25 % w / w, more preferably in an amount in the range from equal to or more than 0.05 % w / w to equal to or less than 15 % w / w, even more preferably in an amount in the range from equal to or more than 0.1 % w / w to equal to or less than 10 % w / w, most preferably in an amount in the range from equal to or more than 0.5 % w / w to equal to or less than 5 % w / w, based on the total weight of the composition. Herein, the total amount may suitably be calculated as the total amount of the combination of any Malassezia restricta postbiotics and any Malassezia globosa present.

[0284]

[0201] Such topical composition preferably further comprises other topical barrier-repair agents, for example one or more compounds selected from the group consisting of ceramides, fatty acids, cholesterol, niacinamide, glycerin, hyaluronic acid, squalane, dimethicone, panthenol, allantoin, urea, shea butter, avocado oil, jojoba oil, almond oil, phospholipids, tocopherol (Vitamin E), vitamin C, bacillus ferment, omega-3 fatty acids, and mixtures thereof.

[0285]

[0202] Further the topical composition may comprise cosmetic excipients, diluents, adjuvants, additives. Examples of cosmetic excipients, diluents, adjuvants, additives as well as active ingredients commonly used in the skin care industry which are suitable for use in the cosmetic compositions of the present invention are for example described in the International Cosmetic Ingredient Dictionary & Handbook by Personal Care Product Council (http: / / www.personalcarecouncil.org / ), accessible by the online INFO BASE (http: / / online.personalcarecouncil.org / jsp / Home.jsp), without being limited thereto.

[0286]

[0203] Preferably the topical composition includes for example panthenol and / or hydroxyacetophenone.

[0287]

[0204] In a preferred embodiment, the topical composition may in addition comprise other actives and / or excipients, such as for example compounds chosen from the group consisting of niacinamide; glycerin; pentavitin; alkanediols; such as propylene glycol, 1 ,2-pentylene glycol, 1 ,2- hexandiol, 1 ,2-heptandiol, 1 ,2-octandiol, caprylyl glycol, glyceryl caprylate, propane-1 ,3-diol and / or propane-1 ,2-diol; fragrances; and / or carriers, such as water and / or glycerol. Further preferences are as exemplified elsewhere in this specification.

[0288]

[0205] The topical composition can advantageously be in the form of a liquid, lotion, cream, foam, scrub, gel, shampoo, conditioner, soap bar or toner, or applied with an implement or via a face or body mask, pad or patch. Non-limiting examples of such compositions include leave-on and / or rinse-off skin lotions and creams, shampoos, conditioners, shower gels, face wash’s, body wash’s, toilet bars, antiperspirants, deodorants, depilatories and sunscreen lotions. Preferred topical compositions according to the invention are facecare and / or bodycare compositions, such as hand creams and / or body lotions.

[0289]

[0206] Herein below the invention is illustrated by the following non-limiting examples.

[0290] Examples

[0291]

[0207] In the below examples 1 and 2, Malassezia postbiotics were produced and compositions comprising Malassezia postbiotics were applied to normal human epidermal keratinocytes (NHEK) under basal conditions (BC), simulating a non-inflamed skin tissue, and under cytokine-mix stimulated conditions, simulating a skin tissue under inflamed conditions. The cytokine-mix stimulated conditions, simulating inflamed conditions, comprise the administering of IL-4 + IL-13 + IL-22 + TNF-a. In the examples, a cytokine mix was applied at 3 nanogram per millilitre (ng / ml).

[0292] Exam le 1 : Method for preparation of Malassezia postbiotics

[0293]

[0208] The Malassezia postbiotics were prepared as follows:

[0294]

[0209] Step 1: Liquid cultures of Malassezia restricta (Example R) Malassezia globosa (Example G), Malassezia sympodialis (Example S) and Malassezia furfur (Example F) were grown in mDixon medium at 32 °C in a shaker until early stationary growth phase. Malassezia cell pellets were collected through centrifugation at 1 ’000 Relative centrifugal force (ref) for 10 minutes and stored at -80 °C. The supernatant (SN), consisting of conditioned medium, was also stored at -80 °C.

[0295]

[0210] Step 2: 1 ml of each harvested Malassezia cell pellets were resuspended in 2 ml ice-cold Phosphate-buffered saline (PBS) with a pH of about 7.4

[0296]

[0211] Step 3: 1 .5 ml of each resuspension were transferred into Lysing Matrix Y. 100 tubes. Lysis of cells was performed with the MP FastPrep-24 5G bead beating grinder and lysis system during 5 rounds of 1 minutes with cooling steps on ice in between the beating to generate cell lysates.

[0297]

[0212] Step 4: The cell lysates were centrifuged at 10’000 ref for 10 minutes at 4 °C, and the first supernatants (1st-SN) were transferred into new sterile 1 .5 ml Eppendorf tubes 4

[0298]

[0213] Step 5: 10 microliter (pl) of 1st-SN was spotted onto mDixon agar plates, which were incubated at 32 °C for 1 week, to verify that the 1st-SN did not contain any life cells, which would contaminate the tissue cultures during planned assays.

[0299]

[0214] Step 6: Conditioned 1st-SN was centrifuged at 10’000 ref for 10 minutes at 4 °C, and secondary supernatant 2nd-SN was transferred into new sterile 1 .5 ml Eppendorf tubes.

[0300]

[0215] Step 7: Relative protein concentrations of lysates were determined by Pierce BCA protein assay kit.

[0301]

[0216] Step 8: Protein concentrations in Malassezia lysates were adjusted to the same level (Without wishing to be bound by any kind of theory it was assumed that protein concentrations correlate with bioactive metabolite concentrations).

[0302]

[0217] Step 9: 1 ml of each Malassezia postbiotic preparations, consisting of 20% 2nd-SN (from step 6.) and 80% lysate (from step 4.) were sent for differential gene expression analysis (see protocols below).

[0303]

[0218] A graphical summary of preparative steps above is provided in Figure 1 .

[0304]

[0219] The Fungal Postbiotics were named F, S, R and G respectively, wherein F, S, R and respectively G referred to the Malassezia species, furfur, sympodialis, restricta and globosa, respectively, from whom the postbiotics were prepared.

[0305]

[0220] The test compounds are summarized in Table 1 below:

[0306] Table 1 : Test compounds

[0307] *Before treatment as outlined below, each solution was centrifugated at +4°C and the supernatant was used for cell treatment.

[0308] Example 2: Assessment of effects of Malassezia postbiotics preparations on gene expression profile of normal human epidermal keratinocytes under basal or cytokine mix- stimulated conditions

[0309]

[0221] Subsequent to example 1 , the effects of Fungal Postbiotic F, S, R and G were investigated in normal human epidermal keratinocytes (NHEK) in example 2.

[0310]

[0222] The cell type applied was the normal human epidermal keratinocytes (NHEK), Reference Bioalternatives K341 , used at the 3rd passage.

[0311]

[0223] Testing was carried out under basal and cytokine mix-stimulated conditions at 37°C and 5% CO2 The culture medium comprised keratinocyte-SFM, optimized for the assay, supplemented with epidermal growth factor and pituitary extract

[0312]

[0224] More specifically, the effects of Fungal Postbiotic F, S, R and G on a specific gene expression profile were evaluated using Real-Time Quantitative Polymerase Chain Reaction (RT- qPCR) technology. Extracted mRNA was analyzed using a PCR array amongst others the following genes: RPS28 (housekeeping gene); GAPDH (housekeeping gene); CCL5; CCL27; and IVL. The housekeeping genes were included as a control.

[0313]

[0225] Prior to the assay, a preliminary cytotoxicity assay was performed using a standard WST- 8 reduction assay, in order to determine the concentrations to be tested in this study. The assay medium included Keratinocyte-SFM optimized for the assay. The cell type in the preliminary cytotoxicity assay was NHEK in assay medium. Incubation time was twice 24 hours, i.e. 24 + 24 hours. Evaluation parameters included WST-8 reduction assay and morphological observations with a microscope. After treatment, the cells were incubated with WST-8 (highly water-soluble tetrazolium salt) reduced in a water-soluble orange coloured product (formazan) by succinate dehydrogenase (mitochondrial enzyme). This transformation was proportional to the number of living cells and their metabolic activity. The optical density (OD) of the extracts at 450 nm was recorded with a spectrometer (VERSAmax, Molecular Devices).

[0314]

[0226] During the assay the culture and treatment were carried out as follows: Keratinocytes were seeded in 24-well plates and cultured in culture medium for 24 hours and in assay medium for a further 24 hours. for basal conditions (simulating a non-inflamed skin) : the medium was then replaced by assay medium containing - or in the case of the control not containing - the test compounds (i.e. Fungal Postbiotic 1-F, 1-S, 1-R and 1-G) and the cells were incubated for 48 hours with a treatment renewal after 24 hours of incubation. for cytokine mix-stimulated conditions (simulating an inflamed skin): the medium was then replaced by assay medium containing - or in the case of the control not containing - the test compounds (i.e. Fungal Postbiotic 1-F, 1-S, 1-R and 1-G) or the reference (JAK Inhibitor I tested at 10 pM) and the cells were pre-incubated for 24 hours. After the pre-incubation, the treatments were renewed, the mix of cytokines (IL-4 + IL-13 + IL-22 + TNF-a, each tested at 3 ng / ml) was added and the cells were then incubated for 24 hours.

[0315]

[0227] The tests under each experimental condition were performed in triplicate (n=3).

[0316]

[0228] At the end of the incubation time, the cells were washed in phosphate buffered saline (PBS) solution and immediately frozen at -80°C.

[0317] Differential expression analysis

[0318]

[0229] In the context of the differential expression analysis the differences in expression of the specific gene markers under the different conditions were assessed. Within the context of this analysis the chemokines IL8 (lnterleukin-8), CCL5 (Chemokine (C-C motif) ligand 5) and CCL27 Chemokine (C-C motif) ligand 27) can serve as gene markers for inflammatory responses.

[0319]

[0230] In this respect it is noted that relative expression is different from absolute expression. Absolute expression counts the exact, absolute number of expressed genes (e.g. 1 mlln copies of the mRNA coding for the specific protein) wherein the exact number of mRNA copies are representative for the total concentration of the resulting protein. Relative expression determines the number of expressed genes relative to a control, for example the gene expression in one sample relative to the gene expression in a control sample.

[0320]

[0231] In the experiments the expression of markers was analyzed using RT-qPCR method on total RNA extracted from the cell monolayers of each experimental condition (before RNA extraction, the replicates of the same experimental condition were pooled).

[0321]

[0232] The analysis of transcripts was performed in duplicate (n=2) using a PCR array targeting multiple genes including the RPS28 gene (housekeeping gene); GAPDH gene (housekeeping gene); CCL5 gene; CCL27 gene; and IVL gene

[0322] RNA extraction and Reverse Transcription

[0323]

[0233] Total RNA was extracted from each sample using TriPure Isolation Reagent® according to the supplier’s instructions.

[0324]

[0234] The quality of RNA was evaluated using capillary electrophoresis (4200 Tapestation System, Agilent technologies). The quantity of RNA was evaluated using a spectrophotometer (Synergy H1 , BioTek Instruments).

[0325]

[0235] The complementary DNA (cDNA) was synthetized by reverse transcription of total RNA in presence of oligo(dT) and « Transcriptor Reverse Transcriptase » (Roche). The cDNA quantities were then adjusted before PCR step.

[0326] Quantitative PCR

[0236] The PCR (Polymerase Chain Reaction) was performed using the « LightCycler® » system (Roche Molecular System Inc.) according to the supplier’s instructions. The reaction mix (10 pl final) was prepared as follows:

[0327] - 2.5 pl of cDNA,

[0328] - primers (forward and reverse),

[0329] - reagent mix (Ozyme) containing taq DNA polymerase, SYBR Green I and MgCh

[0330]

[0237] The incorporation of fluorescence in amplified DNA was continuously measured during the PCR cycles. This resulted in a “fluorescence intensity” versus “PCR cycle” plot allowing the evaluation of a relative expression (RE) value for each marker.

[0331]

[0238] The value selected for RE calculations is the “output point” (Ct) of the fluorescence curve.

[0332]

[0239] The PCR array used included 2 reference genes (GAPDH and RPS28). These housekeeping genes were used for data normalization since their expression is constitutive and theoretically stable. Consequently, the level of expression of the target markers was compared to the mean expression level of these 2 markers for all test conditions.

[0333]

[0240] The results are provided in tables 2, 3, 4 and 5 below. Table 2 illustrates the effect of the cytokine mix conditions on the gene expression profile and shows that when going from the basal / non-inflamed conditions (BC) to the cytokine mix / inflamed conditions (CC) the expression of CCL5 and CCL27 goes up, whilst the expression of IVL goes down. To allow for a standardized interpretation, the interpretation of the results as illustrated in Table 3 was applied. In tables 4a and 4b and tables 5a and 5b the results following application of the Malassezia postbiotics are shown. To allow for a good comparison, in each case the gene expression profile of the control without application of the Malassezia postbiotics, was set at 100.

[0334]

[0001] In the tables BC= under basal conditions; CC= under cytokine mix-stimulated conditions; RE = Relative expression (mean % of control)

[0335] Table 2: Effects of cytokine mix conditions on gene expression profile

[0336] Table 3: Interpretation of results

[0337] Table 4a: Relative expressions of genes under basal conditions (BC), simulating noninflamed skin

[0338] In the table 4a, the (BC> indicates the difference (increase (+) or decrease (-)) in relative expression compared to cells under basal conditions of the control where no Malassezia postbiotics were applied. For each gene, the control value under basal conditions is set at 100.

[0339] Table 4b: Interpretation of the results under basal conditions (BC), simulating noninflamed skin

[0340]

[0241] The results in tables 4a and 4b show no effect or even some stimulation of the inflammation markers CCL5 and CCL27 in the presence of the Malassezia postbiotics. The expression of the IVL gene advantageously increases in the presence of some of the Malassezia postbiotics, allowing for a more robust skin.

[0341] Table 5a: Relative expressions of genes under cytokine mix-stimulated conditions (CC), simulating inflamed skin

[0342] In the table the (cc> indicates the difference (increase (+) or decrease (-)) in relative expression compared to cells under cytokine mix-stimulated conditions of the control where no Malassezia postbiotics were applied. For each gene, the control value under cytokine mix-stimulated conditions is set at 100.

[0343] Table 5b: Interpretation of the results under cytokine mix-stimulated conditions (CC), simulating inflamed skin

[0242] The results in tables 5a and 5b illustrate a reduction in inflammation response in the presence of the Malassezia postbiotics. The molecules within the postbiotics reduce the inflammation markers CCL5 and CCL27. Surprisingly the CCL5 and CCL27 gene expression under the cytokine mix induced inflammation decreases in the presence of Malassezia postbiotics, as compared to the control where no Malassezia postbiotics are present. The IVL gene remains stimulated and stimulation advantageously increased, making the skin more robust.

[0344]

[0243] The invention thus allows for a significant advancement in cosmetic science, offering a holistic approach to combating inflammation and its cumulative impact on skin aging, providing consumers with an effective solution for maintaining a youthful and radiant complexion.

[0345] Example 3 : Assessment of Lipid production

[0346]

[0244] For this example, the effects of compounds F, R, S and G (Malassezia furfur, M. restricta, M. sympodialis and M. globosa postbiotics) were assessed on several parameters related to sebum production.

[0347]

[0245] In the below the following abbreviations are used:

[0348] (compound) F = Malassezia furfur postbiotics;

[0349] (compound) R = Malassezia restricta postbiotics;

[0350] (compound) S = Malassezia sympodialis postbiotics; and

[0351] (compound) G = Malassezia globosa postbiotics.

[0352] Unless explicitly stated otherwise, these were prepared as described above.

[0353]

[0246] More specifically, the effects of these compounds were evaluated on lipid synthesis and storage in SEBO662AR sebocytes cultured in presence of a lipogenic mix with androgen (testosterone)-stimulated conditions after 7 days of culture. The analyses of the lipid droplet formation were performed by fluorescent labeling using a Bodipy® lipid probe and image analysis.

[0354]

[0247] During the differentiation of sebocytes, the competent cell of the sebaceous gland, the metabolic activity was mainly focused on lipid biosynthesis (lipogenesis). These lipids were stored as lipid droplets inside the sebocytes.

[0355]

[0248] An immortalized human sebocyte cell line (SEBO662), which responds to lipogenic stimuli, was used to test potential inhibitors. This cell line was modified in order to express a functional receptor to androgens (AR), as in the sebaceous gland (SEBO662AR cell line). In this model, active androgens (dihydrotestosterone (DHT), testosterone), tested alone in a basal non-supplemented medium, induced strong specific responses, including the expression of androgen sensitive-genes but also the induction of a sebocyte differentiation program and lipid synthesis and storage.

[0356]

[0249] The stimulation of SEBO662AR sebocytes by a complete lipogenic mix including testosterone or DHT but also other lipogenesis inducers more frequently described in the literature for sebocyte models (such as insulin or vitamin D3), resulted in an even stronger increase of the lipid synthesis and storage. In this experimental model, cerulenin, an inhibitor of fatty acid synthesis and steroid metabolism was used as a reference in order to inhibit the effect of lipogenic mix with androgen.

[0357] Experimental protocol:

[0358] Culture and treatments:

[0359]

[0250] The sebocytes were seeded in a 96-well plate and cultured for 24 hours in culture medium. The medium was then replaced by assay medium containing or not (control) the test compounds or the reference (Cerulenin tested at 10 pM) and the cells were pre-incubated for 4 hours. Then, the lipogenic mix with androgen (containing vitamin C, vitamin D3, insulin, calcium and Dihydrotestosterone (DHT)) was added and the cells were incubated for 7 days. At mid-term, i.e. after 3 days of incubation, half of the medium was removed, and the treatments were renewed (including lipogenic mix with androgen stimulation). Non-stimulated control conditions were performed in parallel. All experimental conditions were performed in n=3.

[0360] Lipid content analysis (Bodipy® labeling):

[0361]

[0251] At the end of the incubation, the cells were fixed and permeabilized. The lipid droplets contained in the cells were then labeled using a specific Bodipy® fluorescent lipid probe labeling mainly neutral lipids. In parallel, the cell nuclei were stained using a Hoechst 33258 (bis-benzimide) solution.

[0362]

[0252] The acquisition of the images was performed using ImageXpress Micro Confocal (Molecular Devices). Five photos were taken per well (x20 objective lens). Representative images of controls, reference and active compounds were taken.

[0363]

[0253] The labeling was quantified by fluorescence intensity measurement normalized to the total number of cells (Integration of numerical data with the MetaXpress software, Molecular Devices).

[0364] Results:

[0365] The effect of the compounds F, R, S and G (respectively Malassezia furfur, M. restricta, M. sympodialis and M. globosa postbiotics) on lipid synthesis in SEBO662AR sebocytes under stimulated conditions was illustrated by the results. The results are shown in the table 6 below and in figure 2. In the table :(1)= Threshold for statistical significance; ns= > 0.05, Not significant; * = 0.01 to 0.05, Significant; ** = 0.001 to 0.01 , Very significant; *** = < 0.001 , Extremely significant; RFU= Relative Fluorescence Units.

[0366]

[0254] Under basal non-stimulated conditions (see figure 2, top row, left picture), the lipid droplet formation, analyzed by Bodipy® fluorescent labeling, was limited in SEBO662AR sebocytes. The treatment of SEBO662AR sebocytes with the lipogenic mix with androgen for 7 days clearly and strongly stimulated the formation and the accumulation of lipid droplets (see figure 2, top row, middle picture). The reference cerulenin, tested at 10 pM, displayed a significant inhibition of the lipogenic mix-induced lipid droplet formation (65% of the stimulated control) (see figure 2, top row, right picture). These results were expected and validated this assay.

[0367]

[0255] Under the experimental conditions of the assay, compound F (Malassezia furfur postbiotics), tested at 0.125% and 0.25%, inhibited lipogenic mix containing androgen-induced lipid droplet formation with a similar effect at both concentrations (62% of the stimulated control). Tested at a higher test concentration, the compound also displayed an inhibitory effect (71 % of the stimulated control). (See figure 2, 2nd row below “F”, with left picture at 0.125 %, middle picture at 0.25 % and right picture at 0.5 %)

[0368]

[0256] Compound S had no effect on lipogenic mix containing androgen-induced lipid droplet formation. (Compound S was not included in figure 2)

[0369]

[0257] Both compounds R and G induced a concentration-dependent overstimulation of lipid droplet formation induced by lipogenic mix containing androgen, with up to 199% and 145% of the stimulated control for compounds R (Malassezia restricta postbiotics) and G (Malassezia globosa postbioticsj, respectively. (See figure 2, 3rd row below “R”, with left picture at 0.125 %, middle picture at 0.25 % and right picture at 0.5 %, respectively last (4th) row below “G” with left picture at 0.125 %, middle picture at 0.25 % and right picture at 0.5 %) Conclusions:

[0370]

[0258] Excessive oily skin can often be part of the reasons for development of skin and scalp disorders, such as acne and dandruff, which are associated with overgrowth of lipophilic bacterial and fungal microbes, such as Cutibacterium acnes and Malassezia, respectively. Without wishing to be bound by any kind of theory, oil production by skin is accomplished by sebocytes, forming the sebaceous gland of hair follicles. Malassezia furfur postbiotics were shown to Inhibit oil (sebum) production by sebocytes, and can be consequently used as ingredients in topical formulations for treatment of excessive oily skin and associated skin disorders, such as acne and dandruff. Conversely, Malassezia restricta and Malassezia globosa postbiotics stimulated oil (sebum) production by sebocytes. Consequently, topical formulations containing such ingredients can be used to treat skin with low endogenous sebum production, often observed in dry, postmenopausal skin.

[0371] Table 6: Tests under stimulated conditions ((Lipoqenic mix with androgen) and non-stimulated control

[0372] (1)= Threshold for statistical significance; ns= > 0.05, Not significant; * = 0.01 to 0.05, Significant; ** = 0.001 to 0.01 , Very significant;

[0373] *** = < 0.001 , Extremely significant; RFU= Relative Fluorescence Units.

Claims

44CLAIMS1. A topical composition, preferably a skincare and / or haircare composition and / or a composition to treat, care for or improve the appearance of the skin and / or scalp, more preferably an anti-dandruff composition and / or anti-acne composition, wherein such composition comprises one or more Malassezia postbiotics and wherein the Malassezia postbiotics are derived from Malassezia species furfur.

2. The topical composition according to claim 1 , wherein the Malassezia postbiotics is present as an anti-dandruff agent and / or an anti-acne agent.

3. The topical composition according to claim 1 , wherein the composition comprises zinc pyrithione, piroctone olamine, salicylic acid, coal tar, ketoconazole, Climbazole, selenium sulfide and / or Ciclopirox Olamine.

4. A topical composition, preferably a cosmetic skincare composition for non-therapeutic use, wherein such composition comprises one or more Malassezia postbiotics and wherein the Malassezia postbiotics are derived from Malassezia species restricta, Malassezia species globosa or a mixture thereof; and preferably wherein the composition does not comprise Malassezin.

5. The topical composition according to claim 1 , wherein the Malassezia postbiotics is present as an lipid-stimulating agent and / or barrier-repair agent.

6. The topical composition according to any one of the preceding claims, wherein the composition is comprising one or more Malassezia postbiotics, wherein the Malassezia postbiotics comprise a CCL5 inhibitor and / or a CCL27 inhibitor; and / or wherein the Malassezia postbiotics comprise an IVL stimulator.

7. The topical composition according to any one of the preceding claims, wherein the composition is comprising one or more Malassezia postbiotics, wherein the one or more Malassezia postbiotics comprise a Malassezia postbiotic which, when applied to a tissue, preferably a keratinous tissue, preferably under inflamed conditions, results in:- a decrease of relative expression of the CCL5 gene and / or a decrease in concentration of the CCL5 protein, as compared to the tissue wherein the Malassezia postbiotics is not applied; and / or- a decrease of relative expression of the CCL27 gene and / or a decrease in concentration of the CCL27 protein, as compared to the tissue wherein the Malassezia postbiotics is not applied; and / or45- an increase of relative expression of the IVL gene and / or an increase in concentration of the IVL protein, as compared to the tissue wherein the Malassezia postbiotics is not applied.

8. The composition according to any one of the preceding claims, wherein the composition comprises tocopherol, argania spinosa kernel oil, spent grain wax and / or butyrospermum parkii butter extract.

9. The composition according to any one of the preceding claims, wherein the composition further comprises one or more human milk oligosaccharides.

10. The composition according to any one of the preceding claims, wherein the composition in addition comprises a cosmetically acceptable carrier.

11. A process for the preparation of a cosmetic composition, comprising the steps of:- Lysis of one or more Malassezia microorganisms, wherein the one or more Malassezia microorganisms comprise a CCL5 inhibitor and / or a CCL27 inhibitor and / or an IVL stimulator, to yield Malassezia postbiotics; and- Combining the Malassezia postbiotics with a cosmetically acceptable carrier and optionally one or more human milk oligosaccharides.

12. A method of or for the reduction and / or mitigation of inflammation and / or inflammaging and / or the visible effects of inflammation and / or inflammaging, in a tissue, preferably a keratinous tissue such as the skin or scalp, wherein such method comprises the step of application of Malassezia postbiotics and / or of the composition according to any one of claims 1 to 10.

13. The method according to claim 12, wherein the method is a cosmetic and / or non- therapeutic method, preferably a cosmetic method for the treatment of dandruff.

14. A use of or for the reduction and / or mitigation of inflammation and / or inflammaging and / or the visible effects of inflammation and / or inflammaging, in a tissue, preferably a keratinous tissue such as the skin or scalp, wherein such use comprises the step of application of Malassezia postbiotics and / or of the composition according to any one of claims 1 to 10.

15. The use according to claim 14, wherein the use is a cosmetic and / or non-therapeutic method, preferably- a cosmetic use for the treatment of dandruff; and / or- a cosmetic use for the treatment of acne; and / or- a cosmetic use for stimulating lipid production.

Images