WO2024121383A1

WO2024121383A1 - Novel compositions

Info

Publication number: WO2024121383A1
Application number: PCT/EP2023/084886
Authority: WO
Inventors: Riccardo SFRISO; Aline HUEBER; Remo CAMPICHE; Beatrice TASSONE
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2022-12-09
Filing date: 2023-12-08
Publication date: 2024-06-13
Anticipated expiration: 2025-06-09

Abstract

The present invention relates to novel topical compositions comprising postbiotics obtained from Paenibacillus lautus and the use of such compositions in balancing the skin microbiome.

Description

Novel compositions

The human skin is the biggest organ of the human body, and it is the barrier that separates the body from the outer environment. The surface of the skin is colonized by millions of microorganisms, also known as the skin microbiome. Said microbiome includes all kinds of microbiota such as e.g. bacteria, fungi, viruses, archaea, mites (for review, see Byrd et al., Nature Reviews Microbiology, vol.16, 2018, 143-155).

Many extrinsic and intrinsic factors can lead to an imbalance in the skin microbiome or microbiota dysbiosis. It has been shown that through the application of topical antibiotics the skin bacteria could be altered for several days, such that e.g. pathogenic Staphylococcus aureus populations were increased as compared to a decrease in the commensal Staphylococcus ssp population (SanMiguel et al., Antimicrobial Agents and Chemotherap. 2017; Aug 24; 6l(9):e0074-17). Thus, said compounds generally do not selectively act only against the unwanted microbe(s) but often also lead to the removal of the beneficial ones, which in turn may lead to dysbiosis and thus a weakened skin barrier resulting in adverse skin reactions which is not desired.

To keep skin homeostasis, the commensal microbiota are in constant interaction amongst each other as well as with the immune system of the host organism to fight against pathogenic microbes, such as e.g. Staphylococcus aureus.

Thus, the promotion of the self-defense mechanism of the skin to foster the maintenance of healthy skin with selective blockage of pathogenic microbes is an ongoing task, whereby negative side-effects as known for topical antibiotics such as e.g. weakening of the skin barrier as well as the decrease in beneficial skin microbes should be minimized.

Surprisingly, it has now been found that certain postbiotics topically applied, particularly postbiotics from Paenibacillus lautus, can lead to a decrease in pathogenic microbes without negatively effecting the skin barrier, such as particularly reducing (decreasing) pathogenic species selected from the strains of Staphylococcus aureus.

Particularly, the present invention is directed to said postbiotics in dried or liquid form and compositions comprising said postbiotics as well as for the use thereof for the preparation of cosmetic, particularly topical, compositions for improvement/ maintenance of a healthy skin barrier, protection of commensal skin microflora and prevention of detrimental effects, such as e.g. inflammation, as associated with skin diseases including but not limited to psoriasis, atopic dermatitis and allergy, as well as non disease-related conditions, such as skin itching, skin aging, particularly improvement/ maintenance of a healthy skin barrier, protection of commensal skin microflora and prevention of negative effects as associated with e.g. psoriasis, atopic dermatitis, cellular apoptosis, itching, skin allergy, as induced by pathogenic microbiota such as e.g. Staphylococcus aureus.

Thus, in a first embodiment, the present invention is directed to a postbiotic obtained from Paenibacillus lautus.

The term "postbiotic" as used herein and in line with the definition of the International Probiotics Association (ISAPP) (Salminen et al., Nature Reviews Gastraoenterology & Hepatology, 18, 649-667, 2021) is defined as a preparation of inanimate/ non-viable microorganisms, i.e. of Paenibacillus lautus, including its components or metabolites that confer a health benefit to the host when applied to the skin microflora (skin microbiota) as disclosed herein, particularly by reducing (decreasing) pathogenic species such as e.g. strains of Staphylococcus aureus.

The terms "postbiotic from Paenibacillus lautus", "postbiotic derived from Paenibacillus lautus", "postbiotic obtained from Paenibacillus lautus" or "P. lautus postbiotic" are used interchangeably herein, meaning that P. lautus is the starting material (progenitor microorganism) for the postbiotic.

For generation of the P. lautus postbiotic as defined herein, strains of P. lautus can be grown by any method known to the skilled person. The culture conditions of P. lautus can be easily defined, in particular, the P. lautus strain can be grown in a fermenter under suitable culture conditions using media as known in the art (see e.g. https://bacmedia.dsmz.de/ medium/l?bacdive=ll47l). The P. lautus biomass can be recovered from the fermentation broth during the exponential growth phase and/or during the stationary phase, by any methods known in the art including but not limited to centrifugation, extraction, maceration, separation, concentration and/or filtration followed by inactivation of the bacteria. Methods for inactivation of viable microorganism and/or extraction of probiotics are well known to those skilled in the art, see e.g. Salminen et al., supra). Thus, the postbiotic might be released after bacterial lysis at the state of maceration with e.g. ethanol.

In one particular embodiment, the P. lautus postbiotic is a dried extract comprising polar and non-polar components, with about 10% being non-polar and about 90% being polar components, including but not limited to sugars, free amino acids, ions, lipids, particularly with sugar monomers and dimers being in the range of less than 1%, free amino acids in the range of less than 10% and ions in the range of less than 20%, all based on total ingredients in said dried extract.

Thus, the present invention is related to a preparation of non-viable microorganism selected from Paenibacillus lautus in the form of a dried extract, particularly wherein said preparation is used for topical applications, such as used in a topical composition. The skilled person known such compositions that are used in topical applications.

Preferably, said dried extract does not comprise or only comprises minor traces of sugars such as rhamnose, glucose and/or fucose, wherein "minor traces" as defined herein means not more than 0.5% for each sugar based on total ingredients in said dried extract.

In another embodiment, the P. lautus postbiotic is a liquid formulation, comprising the dried extract as defined above dissolved in a suitable solvent, including but not limited to 1,3 propanediol (PDO), glycerol, ethanol, water, preferably PDO. In one particular embodiment, the liquid formulation as defined herein comprises a range of 2 to 5% dried extract in the solvent, particularly a range of 2-5% dried extract in PDO, such as e.g. about 2, 2.5, 2.8, 3, 3.2, 3.5, 3.8, 4, 4.2, 4.5, 4.8 or 5% dried extract in PDO, including chemically produced PDO or biologically-derived PDO.

In a further embodiment, the present invention is directed to a composition, particularly a cosmetic composition, comprising a P. lautus postbiotic as defined herein, such as e.g. the dried extract or the liquid formulation as defined herein, wherein the final concentration of the P. lautus postbiotic in the composition, particularly cosmetic composition, is in the range of 0.001 to 0.01%. As used herein, the term "topical" means external application to keratinous substances, which are in particular the skin, scalp, eyelashes, eyebrows, nails, mucous membranes and hair, preferably the skin.

The term "commensal skin microflora" as used herein includes resident microbiota including but not limited to Staphylococcus epidermidis, Staphylococcus hominis, Cutibacterium acnes, Corynebacterium striatum, Micrococcus luteus, Micrococcus yunnanensis, (see for example Byrd et al. 2018, Nat Rev Microbiol 16:143). A "maintenance or improvement of the commensal skin microflora" includes but is not limited to protection of the commensal skin microflora, keeping or fostering a diverse microbiome free from dysbiosis, and in homeostasis with the skin.

As used herein, "maintenance/improvement of the skin barrier" or "balancing the skin microbiome" or "maintenance or promotion of the skin barrier" are used interchangeably herein and means keeping a healthy skin barrier that protects the organism from excess water loss (low transepidermal water loss), maintain the expression of key markers responsible for a healthy skin barrier, and maintaining a skin microbiome in homeostasis with the skin.

In a further embodiment, the present invention relates to the use of said P. lautus postbiotic or a composition, particularly cosmetic composition, comprising said postbiotic, with a final concentration of at least about 0.001% in said composition, particularly cosmetic composition, as skin protective agent, i.e. an agent which exhibits protective activity on the commensal skin microflora. In particular, the present invention is directed to the use of a postbiotic and/or composition comprising said postbiotic according to the present invention for balancing the skin microbiome, maintenance and improvement of the skin barrier, prevention of detrimental effects to the skin due to colonization of the skin by pathogenic microbes, such as in particular colonization by Staphylococcus aureus.

In one embodiment, the invention relates to a P. lautus postbiotic and/or a composition, particularly a cosmetic composition, comprising said postbiotic as defined herein for improving/ maintaining the self-defense mechanism of the skin, in particular the self-defense mechanism towards pathogenic microbes including but not limited to Staphylococcus aureus, as well as to methods or use of said postbiotic and/or compositions comprising said postbiotic for improving of such skin self-defense mechanism, said method or use comprising contacting the skin with a composition, particularly cosmetic composition, comprising said P. lautus postbiotic as defined herein.

In another embodiment, the invention relates to a P. lautus postbiotic and/or a composition, particularly a cosmetic composition, comprising said postbiotic as defined herein for reducing and/or abolishing cellular skin apoptosis, as well as to methods or use of said postbiotic and/or compositions comprising said postbiotic for reduction and/or abolishing of said cellular skin apoptosis, said method or use comprising contacting the skin with a composition, particularly cosmetic composition, comprising said P. lautus postbiotic as defined herein.

In another embodiment, the invention relates to a P. lautus postbiotic and/or composition, particularly cosmetic composition, comprising said postbiotic as defined herein for improvement and maintenance of commensal skin microflora, in particular wherein the balance is not shifted towards Staphylococcus aureus, as well as to method or use of said postbiotic and/or compositions comprising said postbiotic for maintenance and improvement of said commensal skin microflora, said method or use comprising contacting the skin with a composition, particularly cosmetic composition, comprising said P. lautus postbiotic as defined herein.

In another embodiment, the invention relates to a P. lautus postbiotic and/or composition, particularly cosmetic composition, comprising said postbiotic as defined herein for reducing and/or abolishing colonization or adhesion of pathogenic skin microbes, in particular Staphylococcus aureus, to the skin surface as well as to methods or use of said postbiotic and/or compositions comprising said postbiotic for reduction and/or abolishing of said colonization/adhesion, said method or use comprising contacting the skin with a composition, particularly cosmetic composition, comprising said P. lautus postbiotic as defined herein.

In another embodiment, the invention relates to a P. lautus postbiotic and/or composition, particularly cosmetic composition, comprising said postbiotic as defined herein for reducing and/or abolishing itch sensation and/or inflammation as associated with atopic dermatitis, psoriasis, allergy etc., as well as to method or use of said postbiotic and/or compositions comprising said postbiotic for reduction and/or abolishing of said itching or inflammatory events associated with said diseases.

As used herein, "improving or maintenance of the self-defense mechanism of the skin" means maintaining and fostering a healthy skin barrier, particularly the stratum corneum. In addition, maintaining and fostering the skin innate immunity (e.g. anti-microbial peptides), including keeping a healthy microbiome in homeostasis with the skin.

As used herein, "reducing and/or abolishing of cellular skin apoptosis" means preventing excess apoptosis due to environmental insult, such as e.g. including but not limited to an excess apoptosis due to an increase of pathogenic microbes like Staphylococcus aureus. Apoptosis is commonly measured by counting apoptotic cells and compare the number to a control without these apoptotic events.

As used herein, "reducing and/or abolishing of colonization or adhesion of pathogenic skin microbes" means preventing the excess adhesion of pathobionts, such as but not limited to Staphylococcus aureus, and to maintain a healthy and divers skin microbiome.

The term "skin inflammation" includes a heating and burning sensation accompanied with skin reddening/erythema, on molecular level associated with the increased synthesis and release of inflammatory markers such as but not limited to cytokines, matrix-metalloproteases, and serine proteases.

As used herein, "reducing and/or abolishing of itching or inflammation/skin reddening associated with atopic dermatitis, psoriasis, allergy" and the like means keeping cellular and molecular reactions stimulating or enhancing inflammation and its associated sensations (e.g. itch, burning, stinging) in check or lowering it.

In one embodiment the postbiotic according to the present invention is a liquid formulation consisting essentially of the postbiotic as defined herein in the form of a dried extract and PDO, wherein the percentage of the postbiotic is in the range of 2 to 5% by weight.

The term "consists essentially of" as used according to the present invention means that the total amount of ingredients within the extract, formulation or composition ideally sum up to 100 wt.-%. It is however not excluded that small amounts of impurities or additives may be present in the extract/formulation/composition, with the proviso that the total amount of such impurities is preferably less than about 5 wt.-%, more preferably less than about 3, 2, 1 wt.-% and which are e.g. introduced via the respective raw materials and/or processes used. As used herein, the term "impurities" and "additives" are used interchangeably and refer to co-ingredients within the inventive extract, formulations or compositions and that are present therein in an amount of less than 5 wt.-% based on all ingredients present in said dried extracts, formulations or compositions.

"Prevention" includes not only total prevention but also delaying the onset of, lessening the severity of an incident/symptoms of the condition, and/or increasing the length of time between recurrence of the incidence/condition.

Preferred products in all embodiments of the present invention are cosmetic compositions comprising the postbiotic as described herein. Said compositions preferably comprise water and at least one further agent selected from the group consisting of surfactants, emulsifiers, thickeners and oils.

Thus, in another embodiment, the invention is also directed to cosmetic products/ cosmetic compositions comprising water and at least one agent selected from the group consisting of surfactants, emulsifiers, thickeners and oils, wherein the composition furthermore comprises the P. lautus postbiotic according to the present invention in a final concentration in the range of 0.001%, preferably in a range of between 0.001 to 0.01%, such as e.g. in a range of at least about 0.0095, 0.009, 0.0085, 0.008, 0.0075, 0.007, 0.0065, 0.006, 0.0055, 0.005, 0.0045, 0.004, 0.0035, 0.003, 0.0025, 0.002, 0.015, 0.01% by weight.

It is furthermore advantageous, that the water content in said cosmetic compositions according to the present invention is selected in the range from 30 to 90 wt.-%, from 40 to 90 wt.-%, from 45 to 90 wt.-% or from 50 to 90 wt.-%, based on the total weight of the composition. Further suitable ranges are from 30 to 75 wt.-%, from 30 to 70 wt.-%, from 30 to 60 wt.-% and from 40 to 60 wt.-%.

Advantageously, the P. lautus postbiotic and/or composition comprising said postbiotic according to the present invention can also be used in combination with traditional preservatives to improve the preservative activity thereof.

It is well understood that the use according to the invention is preferably non- therapeutic.

The cosmetic products/ compositions according to the present invention are in particular topically applied to mammalian keratinous tissue such as in particular to human or animal skin or the human or animal scalp and hair.

Thus, in one embodiment the present invention is directed to a P. lautus postbiotic and/or composition comprising said postbiotic as defined herein for the use as topical product, i.e. cosmetic product topically applied to the human or animal keratinous tissues including skin, scalp and hair.

The term "cosmetic product" as used in the present application refers to cosmetic compositions as defined under the 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), 4^th edition, 1992.

Suitable surfactants, emulsifiers, thickeners, and oils for the purpose of the present inventions are all surfactants, emulsifiers, thickeners, and oils commonly used in cosmetic applications and which are e.g. listed 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.

The products according to the present invention are generally prepared by admixing a postbiotic/composition comprising said postbiotic according to the present invention in an amount selected in the range 0.005 to 2 wt.-%, more preferably in the range of about 0.01 to 1 wt.-%, most preferably in the range of about 0.03 to 0.75 wt.-% such as in the range of 0.05 to 0.5 wt.-%, based on the total weight of the product with a suitable carrier.

The cosmetic products according to the present invention preferably further comprise a physiologically acceptable medium, that is to say a medium compatible with keratinous substances, such as the skin, mucosa, and keratinous fibers. In particular, the physiologically acceptable medium is a cosmetically acceptable carrier.

The term "cosmetically acceptable carrier" refers to all carriers and/or excipients and/or diluents conventionally used in cosmetic compositions. As used herein, it can be understood to be a physiologically acceptable medium, i.e. a medium compatible for the intended use such as e.g. with keratinous substances, such as the skin, mucous membranes, and keratinous fibers.

In a particular advantageous embodiment, the carrier consists furthermore of at least about 30 wt.-%, more preferably of at least about 40 wt.-%, most preferably of at least about 45, 50, 60, 70, 80, 90 wt.-% of water, such as in particular a range of from 30 to 90 wt.-%, such as from 40 to 90 wt.-%, from 45 to 90 wt.-% or from 50 to 90 wt.-%, based on the total weight of the composition. Further suitable ranges are from 30 to 75 wt.-%, from 30 to 70 wt.-%, from 30 to 60 wt.-% and from 40 to 60 wt.-%.

Preferably, the cosmetic products/ compositions according to the invention are in the form of a suspension or dispersion in solvents or fatty substances, or alternatively in the form of an emulsion or micro emulsion (in particular of O/W- or W/O-type), PIT-emulsion, nano emulsion, multiple emulsion (e. g. O/W/O- or W/O/W-type), pickering emulsion, hydrogel, lipogel, one- or multiphase solution or vesicular dispersion.

The cosmetic products/ compositions in accordance with the invention can be in the form of a liquid, lotion, a thickened lotion, a gel, a cream, a milk, an ointment or a paste.

The cosmetic products/ compositions according to the invention have a pH in the range of 3-10, preferably in the range of pH of 3-8, most preferred in the range of pH 3.5-7.S. The pH is adjusted by methods known to a person skilled in the art, e.g. by using an acid such as a hydroxy acid including glycolic acid, lactic acid, malic acid, citric acid and tartaric acid or a base such as e.g. sodium or potassium hydroxide or ammonium hydroxide as well as mixtures thereof.

Preferably, in the product/ composition according to the invention said acid, if present, is used in an amount of at lowest about 0.0001 wt.-%, such as e.g. in an amount of about 0.01 to 1 wt.-%, in particular in an amount of about 0.01 to 0.5 wt.-%.

The amount of the cosmetic composition to be applied to the skin is not critical and can easily be adjusted by a person skilled in the art. Preferably the amount is selected in the range of 0.1 to 3 mg/cm² skin, such as preferably in the range of 0.1 to 2 mg/cm²skin and most preferably in the range of 0.5 to 2 mg/cm²skin.

The cosmetic products/ compositions according to the present invention are in particular skin care preparations, functional preparations and/or hair care preparations such as most in particularly skin or hair care preparations.

Thus, in one embodiment the present invention is directed to the use of a P. lautus postbiotic and/or composition comprising said postbiotic as defined herein as skin or hair care preparations, i.e. for the preparation of cosmetical products applied to skin or hair of humans or animals.

Examples of skin care preparations are, in particular, light protective preparations (sun care preparations), anti-ageing preparations, preparations for the treatment of photo-ageing, body oils, body lotions, body gels, treatment creams, skin protection ointments, moisturizing preparations such as moisturizing gels or moisturizing sprays, face and/or body moisturizers, as well as skin lightening preparations.

Preferably in all embodiments of the present invention the skin care preparation is a deodorant, an anti-perspirant, or an anti-acne composition.

Examples of functional preparations are cosmetic compositions containing active ingredients such as hormone preparations, vitamin preparations, vegetable extract preparations, anti-ageing preparations, and/or antimicrobial (antibacterial or antifungal) preparations without being limited thereto.

Examples of hair care preparations which are suitable according to the invention and which may be mentioned are shampoos, hair conditioners (also referred to as hair rinses), hairdressing compositions, hair tonics, hair regenerating compositions, hair lotions, water wave lotions, hair sprays, hair creams, hair gels, hair oils, hair pomades or hair brilliantines. Accordingly, these are always preparations which are applied to the hair and the scalp for a shorter or longer time depending on the actual purpose for which they are used.

If the hair care preparations according to the invention are supplied as shampoos, these can be clear liquids, opaque liquids (with pearly luster effect), in cream form, gel-like or else in powder form or in tablet form, and as aerosols. The surfactant raw materials on which these shampoos are based can be anionic, cationic, nonionic and amphoteric in nature and also be present in combinations of these substances.

In another preferred embodiment, the cosmetic compositions according to the present invention are O/W emulsions, W/O emulsions and/or gels such as shower gels or hair gels.

The following examples are provided to further illustrate the compositions and effects of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.

Examples

Example 1: Influence of Paenibacillus lautus postbiotic on epidthelial differentiation

A consortium of commensal bacteria at 1.1 x 10⁶ CFU/ ml of each bacterium was prepared. The bacteria were Staphylococcus epidermidis, Cutibacterium acnes and Corynebacterium striatum. Labskin (Innovenn Labskin, York, UK) was colonized with 10 CFU of the consortium and incubated at 37°C for 2 hours until Labskin surfaced had dried. Tissues were then treated with various concentrations of a dried extract obtained from Paenibacillus lautus fermentation, i.e. P. lautus postbiotic in the form of a dried extract (mix I with 0.01% (final concentration) of dried extract in PDO or mix II with 0.001% (final concentration) of dried extract in PDO) as indicated in the Tables or control (vehicle=H₂O) and incubated for 24 hours. Then, Labskin was infected with 10² CFU/cm² of Staphylococcus aureus and further incubated at 37°C for 48 hours.

Immunohistochemistry was used to detect Cytokeratin-1, a common marker of epithelial differentiation. The result is shown in Table 1.

Table 1: expression of cytokeratin 1 as detected with immunochemistry. "A (%)" means difference of expression using the vehicle as compared to the expression using mix I or mix II; "S.D." means standard deviation, "a.u." means arbitrary units. For further details see text.

The application of both mix I and mix II, i.e. P. lautus postbiotic in different concentrations, resulted in increased epithelial differentiation of at least about 30% as compared to application of a control (water).

Example 2: Anti-inflammatory effect of Paenibacillus lautus postbiotic

Labskin experiments were performed as described in Ex. 1. ELISA (Human Quantikine®) systems were used to detect interleukins IL-8, a common marker to monitor the therapeutic effect in atopic dermatitis patients, and IL-23, a common marker connected to psoriasis, wherein both markers are linked to inflammation.

Table 2A: expression of IL-8 as detected with ELISA. "A (%)" means difference of expression using the vehicle as compared to the expression using mix I or mix II; "S.D." means standard deviation. For further details see text.

The application of both mix I and mix II, i.e. P. lautus postbiotic in different concentrations, resulted in reduced inflammation, measured via reduced expression of inflammation marker IL-8, by between 14 to about 27% as compared to application of a control (water).

Table 2B: expression of IL-23 as detected with ELISA. "A (%)" means difference of expression using the vehicle as compared to the expression using mix I; "S.D." means standard deviation. For further details see text.

The application of mix I, i.e. P. lautus postbiotic in a final concentration of about 0.01%, resulted in reduced inflammation, measured via reduced expression of inflammation marker IL-23, by more than 50% as compared to application of a control (water).

Example 3: Effect of Paenibacillus lautus postbiotic on skin barrier and S. aureus adhesion to the skin surface

To measure the inhibitory effect of P. lautus postbiotic on adhesion of pathogenic microbes such as Staphylococcus aureus associated with a filaggrin deficit on the epidermal barrier, reconstructed human epidermis models (RHE, Skinethic, Lyon, France) were first treated with mix I (see Ex. 1) and immediately colonized with S. aureus (10⁶ CFU/tissue (OD600=0.1)) and incubated at 37°C for 24 hours. Tissues were then collected for histological analysis using fluorescence conjugated antibodies against either filaggrin (Sigma, HPA030188) or S. aureus (Abeam, Ab20920). Filaggrin was visualized with HISTOFINE SIMPLE STAIN AP MULTI alkaline phosphatase (NICHIREI BIOSCIENCES, 414262F) and new fuchsin substrate kit as chromogen (NICHIREI BIOSCIENCES, 415161 F). Nuclei were counter-stained with Mayer's hematoxylin. S. aureus was visualized with Alexa Fluor Plus 555 donkey anti-rabbit (Life Technologies, A32794) and nuclei were counter-stained with DAPI solution (Table 3 and 4). Table 3: expression of filaggrin in the human epidermis model. "A (%)" means difference of expression using the vehicle or mix I as compared to the expression on untreated tissue; "S.D." means standard deviation; "untreated" means human epidermis without colonization with S. aureus. For further details see text.

The application of mix I, i.e. P. lautus postbiotic in a final concentration of about 0.01%, resulted in reduced adhesion of S. aureus, measured via increased expression of filaggrin, by more than 100% as compared to application of a control (water).

Apoptotic cells were detected by TUNEL staining. TUNEL staining was performed by In Situ Cell Death Detection Kit (Roche, cat.11684795910) on paraffin sections following producer's instructions. The nuclei were stained with DAPI. Epidermis treated with mix I and S. aureus, the vehicle (=control) and S. aureus, or untreated were compared for either adhesion of S. aureus (Table 4A) or the number of apoptotic cells (Table 4B).

Table 4A: "A (%)" means difference of fluorescence signal indicative for S. aureus adhesion using the vehicle as compared to the expression using mix I; "S.D." means standard deviation. For further details see text.

The application of mix I, i.e. P. lautus postbiotic in a final concentration of about 0.01%, resulted in reduced adhesion of S. aureus by more than 70% as compared to application of a control (water).

Table 4B: apoptotic cells in response to S. aureus colonization on human epidermis as detected with TUNEL staining. "A (%)" means difference of expression using the vehicle as compared to the expression using mix I; "S.D." means standard deviation; "untreated" means human epidermis treated with mix I but without colonization with S. aureus. For further details see text.

The application of mix I, i.e. P. lautus postbiotic in a final concentration of about 0.01%, resulted in reduced apoptotic cells by more than 30% as compared to application of a control (water).

Example 4: Formulations

Cosmetic formulations in the form of gels, emulsions, shampoo, hair cream, lotion, make-up, toner, after-shave, creams, hand sanitizer are created according to methods known in the art. If not indicated otherwise the pH of the formulations is adjusted to pH 3-7.5 as deemed appropriate. In the following formulations the term "NEW INGREDIENT" refers to Paenibacillus lautus postbiotic, i.e. mix I and mix II in the final concentration as indicated in Table 1.

Table 5: Cosmetic gels comprising P. lautus postbiotic. For further details see Example 1.

Table 6: 0/W emulsions comprising P. lautus postbiotic, Palmitoyl Oligopeptide & Palmitoyl Tetrapeptide-7 are given in [ppm]. For further details see text.

Table 7: 0/W emulsions comprising P. lautus postbiotic. Palmitoyl Pentapeptide- 4 is given in [ppm]. For further details see text.

Table 8: W/0 emulsions comprising P. lautus postbiotic. Palmitoyl Tripeptide-38 is given in [ppm]. For further details see text.

Table 9: 0/W emulsion comprising P. lautus postbiotic for facial cream. For further details see text.

Table 10: natural lotion comprising P. lautus postbiotic. For further details see text.

Table 11: liquid make-up comprising P. lautus postbiotic. For further details see text.

Table 12: moisturizing toner comprising P. lautus postbiotic. For further details see text.

Table 13: after shave SPF 15 comprising P. lautus postbiotic. For further details see text.

Table 14: anti-aging serum comprising P. lautus postbiotic. For further details see text.

Table 15: anti-aging cream comprising P. lautus postbiotic. For further details see text.

Claims

1. A postbiotic originated from Paenibacillus lautus, preferably in the form of a dried extract.

2. A preparation of non-viable microorganism selected from Paenibacillus lautus in the form of a dried extract.

3. Preparation according to claim 2 for use in a topical composition.

4. A liquid formulation comprising the postbiotic of claim 1 or the preparation of claims 2 or 3, wherein the amount of postbiotic or preparation in the formulation is about 2 to 5 wt.-% based on total ingredients in said formulation.

5. Liquid formulation of claim 4 comprising 1,3 propanediol (PDO).

6. A composition comprising the postbiotic of claim 1, the preparation of claims 2 or 3 or the liquid formulation according to claim 4 or 5.

7. Composition of claim 6 for use in a cosmetic application.

8. Composition according to claim 6 or 7, preferably cosmetic composition, wherein the amount of the composition is selected in the range of at least about 0.001 wt.-%, preferably in a range of between 0.001 to 0.01 wt.-%, such as e.g. in a range of at least about 0.0095, 0.009, 0.0085, 0.008, 0.0075, 0.007, 0.0065, 0.006, 0.0055, 0.005, 0.0045, 0.004, 0.0035, 0.003, 0.0025, 0.002, 0.015, 0.01 wt.-% based on total ingredients in said composition.

9. Postbiotic, Preparation or composition according to any of claims 1, 2, 3, 6, 7, 8 for use and/or the preparation of a cosmetic composition for the maintenance or promotion of a healthy skin barrier and/or for improving the self-defense mechanism of the skin and/or for reduction or abolishment of cellular skin apoptosis and/or for maintenance and improvement of commensal skin microflora.

10. Postbiotic, preparation or composition according to any of claims 1, 2, 3, 6, 7, 8 for use and/or preparation of a cosmetic composition against adhesion of pathogenic microbes on the skin surface, particularly adhesion of Staphylococcus aureus, and/or for reduction or abolishment of skin inflammation.

11. Postbiotic, preparation or composition according to any of claims 1, 2, 3, 6, 7, 8 for use and/or preparation of a cosmetic composition for prevention or treatment of itching sensation, preferably itching associated with atopic dermatitis, psoriasis, allergy.

12. Method or process for promoting or maintenance of a healthy skin barrier, improvement of the self-defense mechanism of the skin, reduction or abolishment of cellular skin apoptosis, maintenance and improvement of commensal skin microflora, reduction or abolishment of adhesion of pathogenic microbes on the skin surface, reduction or abolishment of skin reddening, prevention or treatment of itching sensation, said method or process comprising contacting the skin with a cosmetic composition comprising the postbiotic or the preparation of any of claims 1 to 3.

13. Method or process according to claim 12, wherein the cosmetic composition is a topical composition.

14. Method or process according to claim 12 or 13, wherein the cosmetic composition is selected from the group consisting of a sun care product, anti- ageing product, anti-acne product.