WO2024083856A1 - Formulation and device comprising probiotic bacteria strains and uses thereof for pharmaceutical and cosmetic treatment of skin and mucosa disorders - Google Patents

Abstract

The present invention concerns a two-phase formulation comprising (i) a primary powder phase comprising a still alive strain of probiotic bacteria in a freeze-dried state and at least one gelifying agent, and (ii) a complementary aqueous and sterile phase, physically separated from the primary powder phase. The present invention also concerns a device to hold this two-phase formulation, a method for preparing an active probiotic composition therefrom, the active probiotic composition thus prepared and its cosmetic or pharmaceutical uses.

Description

FORMULATION AND DEVICE COMPRISING PROBIOTIC BACTERIA STRAINS AND USES

THEREOF FOR PHARMACEUTICAL AND COSMETIC TREATMENT OF SKIN AND MUCOSA DISORDERS

DESCRIPTION

TECHNICAL FIELD

The invention refers to the general field of topical application of alive probiotic micro-organisms onto healthy skin and to subjects affected by skin disorders, such as acne vulgaris, eczema and atopic dermatitis in particular for restoring the balance in microbial communities and competing with opportunistic bacteria.

The present invention proposes a two-phase formulation comprising two phases physically separated from one another with a primary powder phase comprising probiotic bacteria and at least one gelifying agent, and a complementary aqueous and sterile phase. The present invention also proposes a device to hold this two-phase formulation, a method for preparing an active probiotic composition therefrom, the active probiotic composition thus prepared and its potency in cosmetic and pharmaceutical product uses. The probiotic bacteria in the form of this active probiotic composition are used for topical administration to the skin, scalp and mucosa wherein treatment may involve the co-administration by oral supplementation.

BACKGROUND OF THE INVENTION

The microbiota plays an important role in a wide variety of skin and mucosa disorders. Skin disorders are accompanied with an alteration of the skin microbiota but also with an alteration of the gut microbiota. The microbiota is a key regulator for the immune system as it aims to maintain homeostasis and to communicate with tissues and organs in a bidirectional manner. Dysbiosis, a state of microbial imbalance, has the potential to negatively impact the skin microbiota and its function. Any imbalance in the skin microbiota results in skin disorders. Acne, atopic dermatitis, psoriasis and rosacea are some common skin conditions that arise due to an imbalance in the existing skin microbiota (Navarro-Lopez et al, 2021).

Acne vulgaris (acne) is a common cutaneous disorder that can harm mental health and lead to problems such as anxiety, depression and low self-esteem as the scars prevail during adulthood. This skin condition is present in 85% of adolescents but recent studies have found its presence in children and adults. Skin pathologies such as acne are associated with overgrowth of opportunistic bacteria and require continuous, intensive treatments which may affect the overall life quality of patients. Several external and internal factors, such as diet and hormonal changes may play specific roles. However, the key players of the immunological responses and skin defenses are the commensal bacteria (Yu et al, 2020), which are part of the skin microbiota. The imbalance in the microbial communities results in a colonization by opportunistic bacteria.

Conventional treatments of acne rely on the prolonged use of topical corticosteroids and systemic antibiotics. Treatment aims at inhibiting bacterial colonization such as Cutibacterium acnes, (C. acnes), but this non-specific eradication results in the emergence of bacterial resistance. Other treatments like corticosteroid applications result in skin thinning, rosacea and melanocytes inhibition which affects the treatment benefits.

This has led to a new insight into the utilization of probiotics for improvement of skin health and restoration of skin microbial balance. Probiotics have been defined as "live microorganisms which when administrated in adequate amounts confer a beneficial health effect on the host" as stated by FAO/WHO. Thus, there is a strong need for probiotics that have the capacity to compete with specific bacteria that colonize the follicles of the skin. The skin barrier intervention takes in contest physical and chemical barrier of the stratum corneum, induces a barrier that provides defense against colonization or invasion of microbial pathogens.

Lactiplantibacillus plantarum (L. plantarum) (Zheng et al, 2020), previously known as Lactobacillus plantarum is a subgroup of lactic acid bacteria present in a range of environments, including gastrointestinal tract of mammal, vaginal mucosa and food matrices. L. plantarum produces active substances like bacteriocin-like substances that inhibit toxic bacteria. Experimental evidence highlights the probiotic character of several strains (Gheziel et al, 2019) some of which are commercialized as health-promoting supplements and functional food products (Seddik et al, 2017). The topical application of L. plantarum on severe human burns has been described to improve tissue repair and prevent infections (Peral et al, 2009, Aybar et al, 2022). From this perspective, L. plantarum, represents a good model to deepen the nature of antimicrobials as key factors related to probiotic action of lactobacilli which is beneficial to health.

The action of the probiotic is more than just this beneficial health effect, the definition of probiotic lies in the fact that the microorganism is still alive. If not, the product only meets the definition of a postbiotic. Indeed, a postbiotic is a "preparation of inanimate microorganisms and/or their components that confers a health benefit on the host". But the complexity of generating probiotics which are still alive on the skin and active has been challenging. Therefore, the term probiotic is increasingly found in patent literature and commercial products even if sometimes inaccurately used. No studies have shown survival rate of alive bacteria on the skin. Indeed, because of this strong difficulty, some treatments comprising interesting bacteria such as L. plantarum only refer to "Lactiplantibacillus extract” and not to probiotics because an extraction step inevitably kills a micro-organism.

Also, many topical creams currently in the market use preservatives to prolong their shelf-life but can eventually work by killing both harmful and beneficial bacteria and damaging the skin microbiota once applied topically. As a result, prolonged exposure to preservatives can have detrimental effects on the skin and mucosal health, leading to imbalances, sensitivities, and other unwanted skin conditions. This means that the probiotics are inactivated, and those preservatives cause often more harm than good. For instance, the patent application EP2364712 A1 discloses the stabilization of a formulation thanks to petrochemicals and mineral oils such as petrolatum, paraffin, synthetic wax, which can damage the skin as it can clog the pores and affect the skin barrier.

Along the same lines, current topical formulations are based on the use of postbiotics, obtained through bacterial lysates (dead bacteria) and soluble bacterial metabolites. The postbiotics contain inactivated microbial cells or cell components with or without metabolites. Clinical use of postbiotics has been limited by issues of delivery and formulation and these issues have not been fully addressed.

Other products succeed to produce a formula with an active and alive microorganism but fail to sustain this activity or viability for a long time after the storage of the product or once applied on the skin. Therefore, there is a need to propose truly alive and active probiotics with a strong longevity after storage and after application onto the skin.

The International application WO 2009/133519 A2 discloses a lyophilizate containing at least one live or inactivated, physiologically acceptable microorganism, one of its metabolites or one of its fractions and at least one surfactant with an HLB greater than or equal to 12. This lyophilizate disclosed may be used to prepare extemporaneous cosmetic or dermatologic care product for a keratin material. To that end, a second composition containing a physiologically acceptable medium capable of solubilizing or dispersing this lyophilizate is implemented (page 35, lines 25-28).This second composition may be water such as a bath or a shower in which lyophilizate is directly immerged (page 23, lines 18-19 and page 35, lines 6-9).

The patent application FR 2 913 337 Al implements a) a lyophilized form of at least one physiologically acceptable microorganism, live or inactivated, one of its metabolites or one of its fractions and b) a physiologically acceptable medium, distinct from said lyophilized form to prepare a cosmetic composition for keratin material. When this physiologically acceptable medium is water, it is the water of a shower or a bath or a thermal spring water (page 14, lines 16-25). In example 1, the composition of the formula before lyophilization consists of Lactobacillus paracasei, sorbitol, starch, sodium alginate and water.

The International application WO 2020/127637 Al aims to propose a topical composition comprising viable micro-organisms and implements, to this extend, a microcapsule comprising a fat-based coating surrounding a composition comprising a viable microorganism. The patent application CN 112226429 A also implements a probiotic microcapsules or freeze-dried probiotic microcapsules for use as a cosmetic ingredient. The International application WO 2020/007906 Al proposes an aqueous topical composition prepared by adding a dry composition of viable bacteria and at least one buffer to a water miscible solvent comprising at least one thickening agent. As explained from page 10, line 32 to page 11, line 5, the presence of a buffer is mandatory to maintain the pH in an acceptable range while using an acid or a base to control the viscosity of the water miscible solvent.

None of the documents chosen from the International application WO 2009/133519 A2, the patent application FR 2 913 337 Al, the International application WO 2020/127637 Al, the patent application CN 112226429 A and the International application WO 2020/007906 Al provides any data showing that, when using alive microorganisms, the latter are still alive on the skin once the topical application containing them has been applied thereon.

Despite the interest for the application of probiotics onto the skin, as suggested above, several attempts have failed to develop and optimize a system for topical application, able to keep alive a probiotic bacterium during an extended period of storage and on the skin for a period long enough to have an effective activity.

There is still a need to achieve this longevity while also achieving a concentration of probiotic high enough to successfully take the ascendancy over pathogenic micro-organism and replace them in hair follicle niches. Another challenge is the development of such a formulation that could also be free of petrochemicals, additives, and conservatives. There is also a need for an extemporaneous preparation to deliver a product specific to the skin damages and which can be easily and immediately used by the customer.

SUMMARY OF INVENTION

The invention offers a solution to the challenges stated above. The object of this invention is a two-phase formulation with exclusively natural components, combined to a sealed device that can be easily used by a customer to combine the two phases of the formulation in order to prepare a cosmetic or pharmaceutical composition for a topical application.

Combined to this device, the two-phase formulation succeeds to maintain alive the probiotic bacteria such as lactic acid bacteria and, in particular, L. plantarum without any preservatives, additives, or other petrochemicals, in a topical composition such as a topical suspension that is stable over time. In this way, the probiotic bacteria resist to a long period of storage. Indeed, the ingredients of the two-phase formulation have been carefully formulated and strongly optimized to enhance the viability of the probiotic bacteria such as lactic acid bacteria and, in particular, L. plantarum and contains natural components exclusively.

After mixture of the two phases of the formulation and application of the resulting composition onto the skin, the probiotic bacteria are also alive long enough thereon to modulate metabolic activity, to decrease the lipid production in human skin, to have an anti-inflammatory action and a strong capacity to compete with C. acnes such as C. acnes IA in the presence of 5. epidermidis and then to replace them in hair follicles niches.

DETAILED DESCRIPTION OF THE INVENTION

The present invention uses live probiotic bacteria such as lactic acid bacteria, which have been shown to be metabolically flexible and adapting to different environments. The invention enables the survival of bacteria in the powder phase of the formulation, in a liquid resuspended form after mixing the two phases of the formulation and even on the skin itself. According to the invention the bacteria are alive after an extended storage period and up to 48 hours once applied on the skin as evidenced by the topical application of the composition obtained after mixing the two phases of the formulation to skin biopsies. This long-term viability increases the action of the lactic acid bacteria such as L. plantarum, without any damage to the skin.

In the context of the present invention, the terms "alive", "live" and "active" are equivalent and can be used interchangeably. In the present invention, the strain of probiotic bacteria is alive in the powder phase of the formulation, in the composition obtained after mixing the two phases of the formulation and on the skin after applying the obtained composition thereon. An alive strain of probiotic bacteria used in the present invention must be capable of metabolic activities for a normal growth and preferably for a minimum of 1 year in the powder phase (mortality to reach max 65% of the initial load), for a minimum of 7 days and up to 14 days once the two phases of the formulation are mixed (i.e the composition such as the serum) at 25°C/60%RH and for a minimum of 8 hours on the skin. If the strain is not respecting one of the 3 options, it is not considered alive.

The formulation, i.e. both phases of this formulation, is free of preservatives and tests on cellular models of human skin for acne show an improved metabolic activity and a decreased lipid production demonstrating that the formulation is not toxic and improves the damaged skin. Other experimental evidence shows the capability of decreasing the bacterial count of C. acnes such as C. acnes IA and 5. epidermis and the anti-inflammatory activity upon treatment with the composition. The latter was also tested on 125 healthy subjects and no sign of toxicity was observed, confirming that it is safe to use and non-irritable to the skin.

The formulation may include natural enhancers that promote the viability and efficacy of the probiotic bacteria such as lactic acid bacteria and in particular L. plantarum and act directly to treat acne as a solemnly mono-dose (single dose) and/or multi-dose therapeutic approach. This longevity and non-toxicity for the skin is achieved thanks to the combination of a well-studied selection of natural components and a device that can maintain a sterile environment but at the same time stays favorable to the survival of the probiotic bacteria such as lactic acid bacteria and in particular L. plantarum.

In the present application, all the ranges must be interpreted as limits included.

More particularly, the present invention concerns a two-phase formulation comprising: - a primary powder phase comprising at least one still alive strain of probiotic bacteria in a freeze-dried state and at least one gelifying agent, and

- a complementary aqueous and sterile phase, physically separated from the primary powder phase.

The formulation according to the invention corresponds to a formulation of alive probiotic micro-organisms comprising two different elements: a primary powder phase and a complementary aqueous and sterile phase. Those two phases are physically separated from one another i.e. they are in two different compartments or containers. Typically the primary powder phase is in a 1^st compartment or container that comprises at least one still alive strain of probiotic bacteria in a freeze-dried state and at least one gelifying agent. The complementary aqueous and sterile phase is in a 2^nd compartment or container, different from the 1^st compartment or container.

The primary powder phase comprises at least one still alive strain of probiotic bacteria in a freeze-dried state. Any probiotic bacteria known to the one skilled in the art may be used in the present invention. The primary powder phase of the formulation of the invention can comprise a single strain of probiotic bacteria or a mixture of at least two different strains of probiotic bacteria. In particular, the strain(s) of probiotic bacteria implemented in the invention is/are strain(s) of lactic acid bacteria i.e. a lactobacillus strain. More particularly, the strain(s) of probiotic bacteria implemented in the invention belong(s) to the Lactiplantibacilli genus and in particular comprises or is a strain of Lactiplantibacillus plantarum. Even more particularly, the strain(s) of probiotic bacteria implemented in the invention is or comprise any Lactiplantibacillus plantarum strain available from public collections such as DSMZ or ATCC or any Lactiplantibacillus plantarum strain commercially available such as the Lactiplantibacillus plantarum LP01 which is provided in a freeze-dried state by the Probiotical S.p.a., Novara, Italy. Other species of Lactobacillus and/or Bifidobacterium can be used in the invention, such as Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus paracasei, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus salivarius, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium ramosum, Bifidobacterium lactis, and Streptococcus thermophilus.

The primary powder phase comprises an amount of bacterial strain(s) in a freeze-dried state making it possible to obtain, after mixing the two phases of the formulation, an amount of alive bacterial strain(s) ranging from 10⁴ cells/mL to 10¹² cells/mL and, in particular, from 10⁶ cells/mL to 10⁸ cells/mL of final product. The term "cell" is equivalent to the expression "Colony Forming Unit" (CFU) and can be used interchangeably in the present description. In addition, in the present description, the expressions "final product" and "probiotic active composition" and the term "serum" are equivalent and can be used interchangeably.

Techniques to prepare the bacterial strain(s) in a freeze-dried state such as lyophilization are well-known techniques to the one skilled in the art.

In the two-phase formulation of the present invention, the bacterial strain(s) in a freeze-dried state as previously defined is/are present in a maximum amount by weight of 35%, and advantageously in a maximum amount by weight of 15% with respect to the total weight of the formulation (i.e. weight of the primary powder phase + weight of the complementary aqueous and sterile phase).

It is clear that the bacterial strain(s) in a freeze-dried state and also the primary powder phase must be microbiologically controlled to avoid any contamination by other microorganisms than the bacterial strain(s) of the formulation. Thus the preparation of bacterial strain(s) in a freeze-dried state and of the primary powder phase may be performed in a clean room.

The primary powder phase also comprises at least one gelifying agent.

As used herein, the expression "gelifying agent" can be replaced by the expression "thickening agent". Such an agent may be any suitable material which can increase the viscosity of a liquid without substantially changing its other properties. Some thickening agents are gelling agents (gellants), forming a gel, by dissolving in a liquid phase as a colloid mixture that forms a weakly cohesive internal structure. Others act as mechanical thixotropic additives with discrete particles adhering or interlocking to resist strain. Others are fatty substances acting as emulsifiers. Advantageously, the "gelifying agent" or "thickening agent" implemented in the present invention forms a gel or a mucilage or an emulsion mass upon contact with an aqueous medium. Formation of a gel or a mucilage or an emulsion mass relieves irritation of mucous membranes by forming a protective layer.

The one skilled in the art knows numerous gelifying agents usable in the formulation of the invention. Those substances include also but are not limited to acacia gum, agar, alginic acid, aliphatic polyesters, ammonium, calcium, sodium, or potassium alginate, arabic gum, attapulgite, bentonite, calcium polycarbophil, polycarbophil and polyacrylic acid, carragenan in various grades and types, ceratonia, cholesterol, copovidone, dextrins, dibutyl phthalate, dibutyl sebacate, diethyl phthalate, dimethyl phthalate, ethylene vinyl acetate, gelatin, glyceryl behenate, guar gum, hectorite, inulin, mucin, saponite, shellac, hyaluronic acid and its salts, pectin, poloxamers, polydextrose, polyethylene oxide, polyquaterniums, polyvinyl acetate, propylene glycol alginate, povidone, sulfobutyl ether p-cyclodextrin, tragacanth, trehalose, triacetin, xanthan gum, aluminum monostearate, cetostearyl alcohol, cetyl alcohol, stearyl alcohol, glyceryl monooleate, glyceryl palmitostearate, polyoxyethylene castor oil derivatives, polyoxyethylene stearates, polyoxyl glycerides and sorbitan fatty acid ester and combinations thereof.

In particular, the gelifying agent in the formulation of the invention is a natural gelifying agent and more particularly is hyaluronic acid which is an active ingredient of the formulation. Indeed, L. plantarum accelerates production of skin-health enhancing hyaluronic acid (HA) (Nakai et al, 2016). HA restores hydration, tends to prevent water loss and hence improve skin moisturization. HA is a glycosaminoglycan, a very long chain of disaccharides present in connective tissues that is responsible for retaining moisture. HA is also a substance that attaches to collagen and elastin to form cartilage.

Gelifying agent or thickening agent, in percentage between 0.1-20% w/v (weight of gelifying agent or thickening agent with respect to the total volume of the serum), creates a gel or mucilage or emulsion, aiming to the retention and to the active bacteria topical delivery after the mixing of the two phases. In the two-step formulation of the invention, the primary powder phase further comprises at least one sugar.

The sugar(s) implemented in the present invention may belong, but are not limited, to the group of sugar alcohols. Sugar alcohols are carbohydrates with a chemical structure that partially resembles sugar and partially resembles alcohol. More particularly, sugar alcohols are derived from sugars, containing one hydroxyl group (-OH) attached to each carbon atom. As examples of sugar alcohols, one can cite sorbitol, isomalt, mannitol, maltitol, lactitol, xylitol and erythritol. They can also be the hydrogenated forms of mono-(erythritol, xylitol, sorbitol, mannitol), di-(lactitol, isomalt, maltitol) or polysaccharides (maltitol and sorbitol).

In particular, said at least one sugar present in the primary powder phase of the formulation of the invention is mannitol, sorbitol or a mixture thereof. Indeed, L. plantarum have the ability to utilize preferably mannitol or sorbitol. Sorbitol can serve as a sugar substitute. Mannitol is a polyol (sugar alcohol) with indigestibility properties and has been grouped as a prebiotic that feed bacteria to grow.

The amount of sugar(s) in the primary powder phase ranges from 2% up to 80% w/v (weight of sugar(s) with respect to the total volume of the serum).

The two-phase formulation of the invention also comprises a complementary aqueous and sterile phase. This phase has, as solvent, a water-based solvent, thus justifying the designation of aqueous phase. By "water" is meant, in the context of the present invention, tap water, deionised water, distilled water or even ultra-pure water (18.2 MQ). In a particular embodiment, this aqueous phase only comprises water i.e. consists of water. In another embodiment, this aqueous phase comprises water and at least another excipient typically used in the basic formulation of pharmaceutical or cosmetic formulations, such as glycerin, emollients, emulsifiers, dispersing agent, in amounts which are typical for the desired use.

Within the scope of the invention, the aqueous phase is also sterile which means that the water-based solvent and consequently the aqueous phase are sterile. To reach this goal, the water-based solvent and/or the aqueous phase may have been submitted to a sterilization step such as an autoclaving step or a filtration step or a gamma radiation step. In a particular embodiment, the aqueous and sterile phase of the two-phase formulation comprises or consists of sterile water.

Typically, the complementary aqueous and sterile phase of the formulation according to the present invention is free of preservative, free of surfactant, free of buffer, free of fat and free of gelifying agent.

In addition, in the two-phase formulation of the invention, the primary powder phase and/or the complementary aqueous and sterile phase may further comprise at least one vitamin. In particular, said at least one vitamin is comprised in the primary powder phase.

When the two phases of the formulation comprise at least one vitamin, the vitamin(s) present in the primary powder phase may be identical to or different from the one(s) present in the complementary aqueous and sterile phase.

Advantageously, the vitamin(s) implemented in the two-phase formulation of the invention belong to the B-group vitamins and to salts thereof.

The B-group (or B-complex) vitamins include thiamine (vitamin Bl), riboflavin (vitamin B2), niacin (vitamin B3), pyridoxine (vitamin B6), pantothenic acid (vitamin B5), biotin (vitamin B7 or H), folate (vitamin B11-B9) and cobalamin (vitamin B12). Each B- group vitamin is chemically different and acts in synergy to maintain the body homeostasis for skin.

Vitamin Bl (thiamine), is an essential micronutrient that is involved in energy production and its beneficial for the improvement of acne-prone or dry skin and improves the appearance of fine lines. It also boosts collagen production and aids in skin revitalization. Accordingly, said at least one vitamin implemented in the formulation of the invention is vitamin Bl or a salt thereof such as, for example, thiamine mononitrate.

The amount of vitamin(s) in the primary powder phase and/or in the complementary aqueous and sterile phase ranges from 0.3% up to 7% w/v (weight of vitamin(s) with respect to the total volume of the serum).

Typically, the powder phase of the formulation according to the present invention is free of preservative, free of surfactant, free of buffer and free of fat. In a particular embodiment, the powder phase of the formulation according to the present invention consists of at least one bacterial strain in a freeze-dried state as previously defined, at least one gelifying agent as previously defined, at least one sugar as previously defined and at least one vitamin as previously defined.

The present invention also concerns a device in which the two-phase formulation can be stored before mixing said phases in order to prepare an active probiotic composition. Indeed the sterile freeze-dried probiotic bacteria which are dormant i.e. not proliferating become active upon rehydration with the aqueous and sterile phase. When active, the probiotic bacteria are able to grow and to start their metabolic activity. This metabolic activity can be observed following the pH of the probiotic composition and its diminution and/or the increase in the numbers of CFU.

More particularly, the present invention concerns a device for the preparation of an active probiotic composition, comprising:

- a first container containing the primary powder phase as previously defined, and

- a second container containing the complementary aqueous and sterile phase as previously defined, the first container and the second container being configured to be connected to each other and to allow mixing of these two phases to prepare an active probiotic composition.

In the device according to the present invention, the second container containing the complementary aqueous and sterile phase as previously defined is sterile. The introduction in the first container of the primary powder phase as previously defined is microbiologically controlled and advantageously performed in a clean room and in aseptic conditions to avoid contamination by micro-organisms other than the strain(s) of interest.

Any device implementing two containers that can be connected to each other in order to mix the solid or liquid compositions they contain can be usable in the present invention. In a particular embodiment of the invention, at least one of the first container and the second container is a syringe 1, 10. In another particular embodiment of the invention, at least one of the first container and the second container is a vial 2, a pouch or a capsule.

According to these embodiments, both containers can be (i) syringes connectable to one another or (ii) vials, pouches or capsules connectable to one another or even (iii) one of the containers can be a syringe and the other one a vial, a pouch or a capsule. Figure 1A is a photograph of the device according to the case (iii). In the latter case, the syringe 1 advantageously contains the powder phase and the vial, the pouch or the capsule 2 contains the complementary aqueous and sterile phase. Alternatively, the syringe 1 may contain complementary aqueous and sterile phase and the vial, the pouch or the capsule 2 may contain the powder phase.

The first container and the second container and in particular the syringe(s), the pouch(es) and vial(s) implemented in the invention can be in any non-pyrogenic material typically used in the pharmaceutical or cosmetic domain. This material may be glass or non-pyrogenic plastic. In particular, when at least one of the containers is a vial, the latter may be a glass vial.

In addition, the device of the invention may further comprise a connector 3 configured to connect and seal the first container with the second container in order to avoid any external contamination. This connector may include a channel that provides fluid communication between the first container and the second container. In addition, this connector may present at least one, male or female, luer-lock connection and in particular it may present two luer-lock connections i.e. two male luer-lock connections, two female luer lock connections or one male luer-lock connection and one female luer- lock connection.

In a particular embodiment, the device of the invention may comprise two syringes as the first and the second containers and a connector presenting two luer-lock connections. In this embodiment, both syringes present a male or female luer-lock connection and these luer-lock connections ensure that the first and second syringes may be attached to the connector in an airtight manner and that the first and second syringes may be sealed one to the other one. Figure IB is a photograph of a device according to the invention with two syringes 1, 10 and a connector 3. Figure 1C is a photograph of a device according to the invention with two pouches 100, 110 and a connector 30.

The present invention also concerns a method for preparing an active probiotic composition using the two-phase formulation and the device as previously defined. More particularly, this method comprises the steps of:

- connecting together the first container and the second container of the device as previously defined, and

- adding the complementary aqueous and sterile phase as previously defined contained in this second container to the primary powder phase as previously defined contained in this first container and mixing together both phases, whereby an active probiotic composition is prepared. The mixing step will depend on the nature of the first container and of the second container. In particular, this mixing is performed by applying pressure up and down and/or by shaking.

More particularly, when the first and the second containers are syringes and the mixing is performed by applying pressure up and down. This movement between the two syringes, backwards and forwards, several times, mixes the different phases to a homogeneous active probiotic composition. Alternatively, wherein the first container is a vial or a capsule as previously defined and the second container is a syringe, the mixing is performed by shaking or by pressure.

The method of the invention makes it possible to prepare a final probiotic composition that can be immediately used on the skin, on the scalp or on a mucosa. The daily preparation of this probiotic composition can vary according to the length of therapy or to the visual improvement of the skin or of the mucosa. The device of the invention corresponds to a mono-dose (single dose) or multi-dose applicator given for the total number of days required for the therapeutic or cosmetic intervention based on the degree of severity of the skin disorders.

"Single dose" or "mono-dose" means that is not a probiotic composition that is usable for a long period such as one month. On the contrary, after mixing the two phases and after applicating the probiotic composition, the probiotic composition has to be discarded. Therefore, the device of the invention allows single use topical application.

"Multi-dose" applicator means an applicator comprising several single doses. For example, the multi-dose applicator according to the invention can comprise 30 devices as previously defined for 1 month treatment.

The present invention concerns an active probiotic composition prepared by the method as previously defined.

The active probiotic composition prepared by the method as previously defined is free of preservative, free of buffer, free of surfactant and free of fat. The active probiotic composition as above-defined may be also free of petrochemicals and mineral oils such as petrolatum, paraffin and synthetic wax.

This active probiotic composition consists of a suspension comprising at least one probiotic bacteria strain as previously defined, at least one gelifying agent as previously defined and at least one element selected in the group consisting of sugars as previously defined and vitamins as previously defined.

In particular, this active probiotic composition consists of a suspension comprising at least one probiotic bacteria strain as previously defined, at least one gelifying agent as previously defined, at least one sugar as previously defined and at least one vitamin as previously defined.

More particularly, the active probiotic composition prepared by the method as previously defined is an aqueous suspension consisting of at least one probiotic bacteria strain as previously defined, at least one gelifying agent as previously defined, at least one sugar as previously defined, at least one vitamin as previously defined and water.

In other words, the only components present in this active probiotic composition are at least one probiotic bacteria strain as previously defined, at least one gelifying agent as previously defined, at least one sugar as previously defined, at least one vitamin as previously defined and water. Because of the presence of gelifying agent(s), the active probiotic composition is in the form of a gelled suspension.

In particular, the active probiotic composition comprises at least one probiotic bacteria strain as previously defined in an amount comprised between 10⁴ alive cells and 10¹² alive cells, and in particular between 10⁶ alive cells and 10⁸ alive cells per mL of the composition.

More particularly, this active probiotic composition is suspension and in particular an aqueous suspension comprising or consisting of:

- at least one probiotic bacteria strain as previously defined in an amount comprised between 10⁴ alive cells and 10¹² alive cells, and in particular between 10⁶ alive cells and 10⁸ alive cells per mL of the composition,

- at least one gelifying agent as previously defined in an amount of between 0.5% up to 20% by weight with respect to the total volume of the composition,

- at least one sugar as previously defined in an amount comprised between 2% up to 80% by weight with respect to the total volume of the composition,

- at least one vitamin as previously defined in an amount comprised between 0.3% up to 7% by weight with respect to the total volume of the composition and

- water.

Even more particularly, this active probiotic composition (serum) consists of a suspension and in particular an aqueous suspension comprising or consisting of

- L. plantarum in an amount ranging from 10⁴ alive cells/mL up to 10¹² alive cells/mL, and in particular between 10⁶ alive cells/mL and 10⁸ alive cells/mL of the composition,

- 1.10% w/v of Hyaluronic acid,

- 3.21% w/v of D-mannitol, and

- 0.33% w/v of Bl vitamin as thiamine mononitrate and

- water.

As soon as prepared the active probiotic composition may be deposited on a repository before its topical application on the skin, the scalp or a mucosa of a subject. Such a repository can be a syringe, a spatula, a pipette, a liquid dispenser, a cup, a plate, a bijoux bottle (glass), or a roller. Accordingly, the present invention concerns a kit comprising a device as previously defined and a repository to hold the active probiotic mixture as previously defined and ready to immediately apply on the skin.

As illustrated in the following examples, the mechanism of action of the active probiotic composition of the invention is:

- less production of lipids and increased metabolic activity therewith in the presence of pro-inflammatory mediators;

- prevention and/or reduction of inflammatory responses through antiinflammatory cytokine production and fibronectin interactions, reduction of pro- inflammatory cytokines and

- competition to colonize hair follicles niches, that in acne, are colonized by C. acnes such as C. acnes IA (inflammatory strains) and 5. aureus then, replacement by L. plantarum (probiotic).

Thanks to this mechanism of action, the active probiotic composition of the invention can be used in cosmetic and pharmaceutical (i.e medical) purposes.

Thus, the present invention concerns the active probiotic composition as previously defined for use in preventing and/or treating an epidermis pathology selected in the group consisting of acne, eczema, atopic dermatitis, alopecia, dysbiosis caused by Cutibacterium acne, skin infections caused by Candida auris, vaginal mucosal layer prone to Candida infection and/or by Staphylococcus aureusfor a subject in need, wherein the active probiotic composition is topically applied on the skin, the scalp or on a mucosa of said subject.

In other words, the present invention concerns a method for preventing and/or treating an epidermis pathology selected in the group consisting of acne, eczema, atopic dermatitis, alopecia, dysbiosis caused by Cutibacterium acne, skin infections caused by Candida auris, vaginal mucosal layer prone to Candida infection and/or by Staphylococcus aureusfor a subject in need, consisting in topically applying the active probiotic composition as previously defined on the skin, the scalp or on a mucosa of said subject. The present invention concerns also the cosmetic (i.e. non-therapeutic) use of the active probiotic composition as previously defined for modifying the top skin microbiome and/or for restoring the balance in skin microbial communities and/or restoring an healthy skin microbial communities and/or for restoring skin appearance and/or for improving the aesthetic appearance of damaged area of the skin in a subject suffering of acnes, wherein the active probiotic composition is topically applied on the skin, the scalp or on a mucosa of said subject. In particular, the subject in need may be a subject suffering from acne.

In other words, the present invention concerns a method for modifying the top skin microbiome and/or for restoring the balance in skin microbial communities and/or restoring an healthy skin microbial communities and/or for restoring skin appearance and/or for improving the aesthetic appearance of damaged area of the skin in a subject, consisting in topically applying the active probiotic composition as previously defined on the skin, the scalp or on a mucosa of said subject.

For any subject, the microbial balance is dynamic, maturing with the subject as he/she grows and navigates through the environments around him/her. The skin human microbiome changes over time in response to factors intrinsic and extrinsic to the host.

The balanced level of the microbiological communities used herein means maintaining a surface of the human body e.g. skin, scalp, mucosa, in healthy and infection free condition. Maintenance of a healthy skin microbiome balancing is obtained by way of the topical application of a composition according to the invention and eventually oral supplementation.

The application of a composition according to the invention resulted in a decrease in Cutibacterium acnes (C. acnes) in individuals with acne-prone skin. This decrease in C. acnes is significant because certain strains of C. acnes, specifically C. acnes 1A, are associated with the development of acne. Probiotics, like Lactobacillus plantarum, help to restore balance to the skin microbiome by suppressing the growth of acne- associated C. acnes strains and promoting a more diverse and beneficial microbial community on the skin. This lead to improvements in acne symptoms and overall skin health.

Methods for identifying skin microbiome composition is well know from the art. For example, genomic characterization of bacterial diversity in microbiological communities relies on sequence analysis of the 16S ribosomal RNA gene, which is present in all bacteria and archaea. The 16S rRNA gene contains species-specific hypervariable regions, which allow taxonomic classification, and highly conserved regions, and a binding site for PCR primers. Using current technologies, an organism does not need to be cultured to determine its type by 16S rRNA sequencing.

This topical application may be combined with an oral treatment. Indeed, previous studies have shown that oral supplementation is beneficial for skin amelioration (Kober et al, 2015) and the combination of an oral administration and the topical administration has never been tested before. The composition of such an oral treatment can comprise at least one probiotic bacteria as previously defined and at least one element selected from the group consisting of prebiotic fibers, vitamins such as previously defined, anti-caking agents, aroma, and bulking agents.

The oral treatment composition may comprise one, two, three, four or all of the above additional elements. In a particular embodiment, the oral treatment composition may comprise Bifidobacterium animalis spp lactis such as Bifidobacterium animalis spp lactis BS01, Lactobacillus plantarum such as Lactobacillus plantarum LP01, Lactobacillus rhamnosus such as Lactobacillus rhamnosus LR05; Prebiotic fibers: Fructooligosaccharides (FOS); Vitamin B2; Aroma: Vanilla; Anti-caking agents: magnesium stearate and silicon dioxide; Bulking agent: Maltodextrin. A particular example of an oral treatment composition is given in the below Table 1.

Table 1: Oral treatment composition to be combined with the topical application of the active probiotic composition according to the invention.

The recommendation is to use every day for one month, at least combined with topical application, for instance every 2 days for a total of 15 days of any oral treatment composition as previously defined.

Other characteristics and advantages of the present invention will additionally be apparent to the one skilled in the art on reading the examples below, which are given as an illustration and not a limitation, with reference to the attached figures.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A already presented is a photograph of a device according to the invention comprising a syringe 1 and a vial 2.

Figure IB already presented is a photograph of a device according to the invention comprising two syringes 1, 10 and a connector 3.

Figure 1C already presented is a photograph of a device according to the invention comprising two pouches 100, 110 and a connector 30.

Figure 2 presents the percentage of alive L. plantarum derived from the serum over time normalized by qPCR (Time = 0, 24 h, 48 h, 72 h and 7 days).

Figure 3 presents the percentage of alive L. plantarum derived from the serum ("Formulation ALIVE") and control (L. plantarum only - "Lactobacillus ALIVE") over time (0, 48 h, 7 days, 4 weeks and 12 weeks).

Figure 4 presents the percentage of survival L. plantarum derived from the probiotic serum over time (0, 8h, 24h and 48h) tested on human skin biopsies. Figure 5 presents the survival count of L. plantarum over time from the probiotic serum, tested on fore-head and /or fore-arm. Results are expressed as CFU normalized by logqPCR count derived from skin swabs of 8 healthy subjects after 8 hours topical application of probiotic serum.

Figure 6 presents the top skin microbiome at various conditions tested on skin with genus-level taxonomy indicated. Bar plots show taxa relative abundance at the genus level. LP01_disc: serum on a CuDerm disc (positive control); Skin_alone: Untreated skin sample; Skin_HA: Skin treated with 0.5% hyaluronic acid; skin samples were collected after post probiotic application of serum after Tl=5min: 5 minutes; T2=1H: 1 hour; T3=4H: 4 hours; T5=8H: 8 hours, showing that after the application of the serum the probiotic L. plantarum represents the majority of the bacteria identified

Figure 7 presents the total lipid production assessed in 2D primary human sebocytes tested in control conditions and treatment with probiotic serum.

Figure 8 presents the evolution rate of metabolic activity assessed in 2D primary human sebocytes tested in control conditions and treatment with probiotic serum.

Figure 9 presents the bacterial proliferation of C. acnes IA and 5. epidermidis after 48 hours of incubation with control conditions and probiotic serum treatment.

Figure 10 presents the production of IL-la by ELISA method after 48 hours of incubation with control conditions and serum probiotic treatment.

Figure 11 presents the production of IL-6 by ELISA method after 48 hours of incubation with conditions and serum probiotic treatment.

Figure 12 presents the production of IL-8 by ELISA method after 48 hours of incubation with control condition and the serum probiotic treatment.

Figure 13 presents a patch testing on 25 volunteers for serum topical application.

Figure 14 presents pictures of the skin of patients before and after treatment with the probiotic serum according to the invention. Figure 15 presents the top skin microbiome identified on skin before and after treatment with the probiotic serum according to the invention, with species-level taxonomy indicated. Bar plots show taxa relative abundance at the species level.

Figure 16 presents ex vivo skin models tested for skin penetration studies after topical application of probiotic skin serum and retrieval of appropriate antibody by immunofluorescence technique.

Figure 17 presents the comparison of total viable cell count of Lactobacillus determined as Colony Forming Units per mL product (CFU/mL) for commercial product (YUN product) versus the serum according to the invention in percentages at time 0 and 8h after application, mean of two healthy participants.

EXAMPLES

Example 1: Preparation of the composition according to the invention

Preparation of the primary powder phase:

For a total of 100 mg of the primary powder phase, are mixed together:

- 64.26 mg of D-mannitol,

- 22.00 mg Hyaluronic Acid,

- 6.67 mg Bl vitamin as thiamine mononitrate, and

- 7.07 mg L. plantarum LP01 (range from 10⁴ alive cells/mL up to 10¹² alive cells/mL of the total volume of the serum).

Preparation of the reactivated probiotic composition

The primary powder phase above-prepared is rehydrated with 2 ml of sterile water forming the composition referenced, in the figures and examples, as serum.

Preparation of another reactivated probiotic composition

The primary powder phase above-prepared is rehydrated with 2 ml of sterile water and 86.26 mg of B-complex vitamins.

Example 2: Survival of the L. plantarum As shown in Figure 2, the viability over time 24 h to 7 days shows more than 50% L. plantarum remain alive in the probiotic serum, Viability qPCR by using qPCR- Propidium monoazide (PMA) method was used to quantify alive L. plantarum.

The viability qPCR methodology was used to detect the percentage of alive L. plantarum derived from the serum and control over time 0, 48 h, 7 days, 4 weeks, and 12 weeks (Figure 3). The graph entitled "Formulation ALIVE" shows that the formulation is stable over prolonged period, at 12 weeks 65% of L. plantarum are alive. The L. plantarum on its own (with no natural enhancers) presented in the graph "Lactobacillus ALIVE" shows decreasing percentage of living bacteria for all time-points. In fact an increased number of L. plantarum was observed after 7 days and 4 weeks for the "Formulation ALIVE" when compared to L. plantarum only suggesting an enhanced viability. This enhanced viability might be due to the bacteria utilizing the formula ingredients to support their growth.

The serum was topically applied to skin biopsies and tested over time = 0, 8 h, 24 h and 48 h. To test viability, CFU/mL was counted in technical triplicates and showing the mean for each time-point, expressed in percentage compared to serum, time 0, time8h, time24h and t48h. (Figure 4). From time 8 h to time 48 h, there is a doubling increase of L. plantarum survival which increases steadily over time. This long-term viability increases the action of L. plantarum, without any damages on the skin. Statistical comparison: serum Vs TO, TO Vs T8, TO Vs T24h, TO Vs T48h NS P>0.05; *P<0.05; **P<0.01; ***P<0.001. T-test.

The serum was topically applied on skin (forearm and or fore-head) on 8 healthy volunteers referenced herein as 1 to 8 determining viability counts by qPCR method. Skin swab samples were collected at time 0 (before application) and at time 8h after serum application. Swabs were processed after collection and viable counts were detected by qPCR. The observed value was calculated from standard curve equation in qPCR and presented as Log CFU/mL for the 8 subjects. The dashed line presents the 50% mean LogCFU/mL derived from time 0 in all subjects. In 8 subjects more than 50% mean was observed at time 8h when compared to time 0. Bar graph showing the increased of L. plantarum count over time in logqPCR at 0 and at time 8 hours after serum probiotic application. (Figure 5). Therefore, L. plantarum contained in the serum remained alive up to 8 hours on skin human subjects.

To conclude, the present invention uses living lactic acid bacteria such as L. plantarum, which have shown to be metabolically flexible and adapting to different environments. The invention enables the survival of bacteria in a formula in powder form, in a liquid resuspended form (i.e. serum) and even on the skin itself.

The optimal pH of skin on most of the areas in the skin lies between 4.7 and 5.75, a pH of 7 is considered neutral as that of water. Skin is slightly acidic, and this creates the skin acid mantle that protects the external layer of the skin. The pH of the serum was measured at 8h, 24h and 48h at RT and 4°C. Other parameters were also measured as represented in the table below (Table 2). The pH of the serum at RT and at 4°C ranged from 4.67 to 6.58 which is considered neutral. The optimal pH of the serum is reached after 8 hours of reconstitution (RT) which is equal to 5.6. When the serum is applied overnight, 8 hours total, the pH is ideal for the skin acid mantle.

Table 2: Parameters measured for the serum according to the invention at RT and at 4C°.

Example 3: Top skin microbiome after application of the serum

To study the effect of the serum composition on the top skin microbiome, the composition was topically applied to the subject's skin, 500 pL of serum which corresponded to approximately 8xlO⁷ CFU/mL. Samples were collected using CuDerm discs at different time points, following serum application. Skin microbiota composition was assessed by 16S rRNA gene sequencing, using Nanopore sequencing technology as to identify the skin microbiota composition. Taxonomic abundance is predominantly made of L. plantarum as indicated in Figure 6 . The taxonomic abundance after probiotic skin serum from t=l up to t=5 (8 hours) belongs to the Lactobacillus spp. after application to the subject's skin.

Examples 4-6: Material and Method

In brief human sebocytes derived from facial skin were first seeded in 24 well plates for 3 days before bacteria inoculation. Then, after 48 hours of bacteria inoculation (C. acnes IA and 5. epidermidis), treatments were performed. Each condition was performed in triplicates. Day (-2) is the day that sebocytes were placed in 24 wells plate. Day 0 is the day of sebocytes cell metabolic activity assay using Alamar Blue reagent. Bacteria (C. acnes IA and 5. epidermidis) were inoculated. Day 2 is the end of bacteria incubation. Controls were challenged with lipopolysaccharide (LPS) to induce an antiinflammatory response and serum treatments was applied. Day 3 is the day of treatment renewal without medium change. Day 4 is the end of experiment and start of analysis. A similar cell density was observed in each well to allow for changes to be monitored between different conditions and between any positive or negative controls. Preparation took into account sterility.

Example 4: Effect of the serum composition on 2D primary human sebocytes

In order to study the lipid production, this was assessed in 2D primary human sebocytes from facial skin tested in various control conditions and treated with serum. The total lipids were normalized to the number of cells. The nuclei of cells were labeled, counted and the resulting fluorescence intensity is proportional to the production of the lipids rationalized by the number of nuclei counted. Quantification by fluorometry (TECAN) and microscopic analysis with fluorescence microscope (NIKON).

The results are presented in Figure 7. Conditions tested in 2D human sebocytes for the following: untreated (basal), probiotic serum, linoleic acid (positive control for lipid production), LPS (lipopolysaccharide) that induces an inflammatory challenge. The Serum is applied in the presence of bacteria (C. acnes IA, S. epidermidis) and challenged with LPS. The latter induces an acne skin model in vitro. The in vitro cellular model challenged with LPS in the presence of bacteria and treated with serum significantly reduced total lipid production when compared to untreated and when challenged with LPS. At baseline: in vitro cellular model treated with serum decreased lipid production. T-test compared to untreated (sebocytes) NS P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.001.

In order to study the evolution rate of metabolic activity of human sebocytes, this was assessed in 2D primary human sebocytes tested in various control conditions before and after LPS challenge to induce an acne skin model and treatment with serum. The metabolic activity was measured at day 0 and day 4, before and after treatment using Alamar Blue reagent by fluorescent intensity (Figure 8).

Conditions tested in 2D human sebocytes for the following: untreated (basal), probiotic serum, linoleic acid (positive control for lipid production), LPS (lipopolysaccharide) that induces an anti-inflammatory response. As shown in Figure 8, the serum alone decreased metabolic activity without the presence of an antiinflammatory response; whilst when the in vitro cellular model was challenged with LPS and inoculated with bacteria (C. acnes IA, S. epidermidis) and treated with serum the metabolic activity was increased when compared to bacteria inoculation challenged with LPS and untreated cells. This implies that serum treatment is capable of increasing the metabolic activity of the human sebocytes in an acne skin model as it results in an amelioration of the metabolic performance when compared to basal and conditions challenged with LPS. Metabolic activity also suggests the absence of cell death, reflected as viable cell numbers. This is enhanced with the serum composition with LPS and bacteria compared to other tested conditions. Evolution rate of metabolic activity, T-test untreated (sebocytes) Vs: serum, linoleic acid, bacteria +LPS, bacteria+LPS+serum. *P<0.05; **P<0.01; ***P<0.001, ****P<0.0001.

As a consequence, the serum and thus the composition prepared there from are free of preservatives and tests on cellular models of human skin acne show an improved metabolic activity of the human sebocytes and a decreased lipid production when challenged with an anti-inflammatory stimulus, demonstrating that the composition is not toxic and improves the damaged skin. Example 5: Effect of the Serum on bacterial proliferation

The bacterial proliferation was validated for C. acnes IA (COS agar) and

S.epidermidis (TSA agar) strains after 48 hours of incubation with different control conditions, under specific strains for each condition (Figure 9). Suspensions of ODeoo 0.5 to 1 were made and counted on solid culture medium.

Colony Forming Units (CFU) were used to determine the parameters for preparing the inoculum. Media used: TSA medium: Tryptone Soy Agar: classic isolation medium used for enumeration/isolation of undemanding germs; COS medium: Columbia Blood Agar: a classic isolation medium used for enumeration/isolation of more demanding germs and a classic medium used for anaerobic germs. Conditions tested in 2D human sebocytes for the following: bacteria only, bacteria + probiotic serum. In the presence of bacteria and the serum treatment, this resulted in a decreased bacterial proliferation when compared to bacteria only (inoculum). Serum treatment and inoculation with bacteria resulted in a lower C. acnes IA and S.epidermidis CFU compared to bacteria only (Figure 9).

This experimental evidence shows the capability of the composition (Serum) prepared from the two-phase formulation to decrease the bacterial count of C. acnes and 5. epidermidis which implies that skin microbiome is improved.

Example 6: Effect of the serum on inflammation

An IL-la by ELISA method was used to understand the underlying immune system involvement, pro-inflammatory response. 48 hours after the serum application to the co-culture (sebocytes/bacteria: C. acnes IA and 5. epidermidis), culture supernatants were collected, and interleukins assay was performed. The control conditions tested were for: untreated, serum, bacteria, bacteria + LPS, bacteria+LPS+serum. The serum reduced the levels of IL-1 a when the in vitro human cellular model for acne was challenged with LPS when compared to control: bacteria challenged with LPS and bacteria only (Figure 10). T-test untreated (sebocytes) Vs. serum, bacteria+LPS Vs. bacteria+LPS+serum NS P>0.05; *P<0.05; **P<0.01; ***P<0.001,****P<0.0001 percentage decrease of serum treatment compared to bacteria challenged with LPS.

An IL-6 by ELISA method was used to understand the underlying immune system involvement, inflammatory response. 48 hours after the serum application to the co-culture (sebocytes/bacteria: C.acnes IA and S.epidermidis), culture supernatants were collected, and interleukin assay was performed.

The control conditions tested were for: untreated, serum, bacteria, bacteria + LPS (challenged with LPS), bacteria+LPS+serum. The serum reduced the levels of IL-6 when the in vitro human cellular model for acne was challenged with LPS when compared to control: bacteria challenged with LPS, bacteria only (Figure 11). This implies that serum treatment was capable of decreasing inflammation by not activating cells of the immune system.

T-test untreated (sebocytes) Vs. serum, bacteria+LPS Vs. bacteria+LPS+serum NS P>0.05; *P<0.05; **P<0.01; ***P<0.001, percentage decrease of serum treatment compared to bacteria challenged with LPS.

An IL-8 by ELISA method was used to understand the underlying immune system involvement, inflammatory response. 48 hours after the serum application to the co-culture (sebocytes/bacteria: C. acnes IA and 5. epidermidis), culture supernatants were collected, and interleukin assay was performed.

The control conditions tested were for: untreated, serum, bacteria, bacteria + LPS (challenged with LPS), bacteria+LPS+serum. The serum treatment reduced the levels of IL-8 when the in vitro human cellular model for acne was challenged with LPS when compared to control: bacteria challenged with LPS, bacteria only (Figure 12). This implies that the serum treatment was capable of decreasing inflammation and ameliorate dermatological adverse events which can be induced by LPS.

T-test untreated (sebocytes) Vs. serum, bacteria+LPS Vs. bacteria+LPS+serum

NS P>0.05; *P<0.05; percentage decrease of serum treatment compared to bacteria challenged with LPS. This experimental evidence shows that the composition prepared from the two-phase formulation presents an anti-inflammatory activity.

The composition was also dermatologically tested on 25 healthy subjects and no signs of irritation and/or toxicity was observed (Figure 13).

As a conclusion, the composition includes natural enhancers that promote the viability and efficacy of L. plantarum and act directly to treat acne as a solemnly mono- therapeutic approach. This longevity and non-toxicity for the skin is achieved thanks to the combination of a well-studied selection of natural components and a device that maintains a sterile environment but at the same time stays favorable to the survival of L. plantarum bacteria.

Example 7: Beneficial effects of the serum of the present invention on skin appearance on human subjects

Assessment of the efficacy, the anti-acne potential, the sebo-regulator potential, the clinical and dermatological tolerance of the serum was set up including 100 acne patients with acne-skin defined as grade 2 or 3 on the Global Evaluation acne (GEA) grading scale (18-45 years, all genders). The clinical study has been conducted in the spirit of the European Directives on Good Clinical Practices. Patients were randomized and they applied the serum formulation described above on their facial skin as a single dose, once a day, every day, in the evening for a total of 28 days. The study has been supplemented by taking of standardized photographs and samples of the skin flora by disc (D-squam type) on one pimple at Day 1 and Day 28. Visioface (Courage + Khazaka Electronic GmbH, Cologne, Germany) has been used for taking photographs on DI and D28. It is a complete system that allows facial shots under standard lighting with software allowing the exact repositioning of the subject at different measurement times.

Assessment of the anti-acneic potential was as following: Different acne lesions (comedones/microcysts, papulae, pustules, nodules/cysts) has been counted on the whole face, except the nasal pyramid, on DI and D28 and evaluated by dermatologist. The sum of each total number of acne lesions type, after weighting, has been calculated for each subject at each experimental time and a total acne score has been get. The statistics analysis has been performed by using Software used: IBM SPSS 23 and comparing the number of acne lesions on D28 vs DI using the Wilcoxon test for paired data. The significant threshold has been set at a = 0.05.

The measurement of the excretory activity of the sebaceous glands has been performed by applying a Sebutape®. Sebutape® is an adhesive patch covered with an opaque white film that becomes translucent when in contact with sebum. Sebutape® is applied to skin that has been degreased with modified 70°C alcohol and left in place for 30 minutes. The seboregulator effect has been evaluated by the dermatologist on DI and D28 by scoring the amount of sebum present on patch Sebutape®.

The entire trial has been carried out under specific environmental condition of temperature and relative humidity i.e. ambient temperature has been between 19°C and 24°C and relative humidity between 40 and 60%.

The serum was applied has following:

1 - Hold the syringe upright with the lid on the top. Tap with a finger the top of the syringe lid to remove residual powder from the lid,

2 - Remove the lid and attach the syringe to the adapter by screwing it onto the tip,

3 - Push the assembled syringe onto the glass vial,

4 - Hold the syringe upright, so that the vial with the sterile water will be upside down. Pill the syringe end back and forth about 40 seconds so that the probiotic powder in the syringe mixes well with the sterile water,

5 - Once powder and water are well blended and there are no white powder particles visible, the probiotic will form a serum texture,

6 - Remove the glass vial and adaptor,

7 - Apply on the hand and them directly to facial skin.

Results:

The data of this trial show that, the serum can be safely use (no adverse effect or no toxicity was observed). The majority of the patients included in the study showed a clinical improvement after the application of the serum formulation as exemplified by some picture of the acne spot area of patients before and after topical application of the serum (Figure 14). The serum acts beneficially on acne lesions (comedones, papulae, pustules, nodules) and possesses a seboregulator activity (Table 3). Therefore, overall, those data demonstrate that the serum can act as acne-mitigating agent.

Table 3: Biological effects of the serum on the skin of subjects suffering from acne

Example 8: Skin microbiome modulation with live lactobacilli

Skin microbiome modulation with live lactobacilli was set up including 100 acne patients with acne-skin defined as grade 2 or 3 on the Global Evaluation acne (GEA) grading scale (18-45 years, all genders). Clinical trial and topical application of the serum has been conducted as described in example 7. Thus the treatment consists of a single dose of serum, once a day and each day in the evening for a total of 28 days. The facial skin microbiome was monitored by 16S amplicon sequencing at 2 different points before and after treatment with the serum of the present invention (28 days of the intervention).

Relative abundance of the microbial community during the study period with species level taxonomy is indicated in Figure 15. Top relative abundance resulting from the application of the serum formulation reveals a significant decrease in the taxa of Cutibacterium acnes. Viability is necessary for the L. plantarum (contained in the serum) to inhibit the colonization of pathogenic microbes and to remodel the health-associated skin commensal bacteria. No major impact was noted on the relative abundance of Staphylococcus epidermidis taxa. L. plantarum was significantly present in the microbial community after serum probiotic application.

Example 9: Determination of penetration depth of the formulation serum into the epidermis and dermis layers of the skin by using ex vivo skin biopsy.

Ex vivo skin models were produced (which were surgical waste), transported under optimal conditions to ensure and maintain tissue viability and with certification (AC-2018-3243 and DC-2018-3242) by the French Ministry in charge of research for the preparation and conservation of elements derived from the human body. The ex vivo skin models were tested with topical treatment and injection with formulation serum. Staining with antibody specifically for Lactobacillus was performed for immunofluorescence.

A background in the keratinocytes of the suprabasal layers can be observed in Figure 16, shown as green background staining. In the dermis, there is no positive signal for L. plantarum when topically applied. Injection of L. plantarum is only positive in the area of injection and absent in the surrounding area. The probiotic serum (containing L. plantarum) does not penetrate in the dermis topically and its surroundings when injected in the ex vivo skin models.

In conclusion, the serum formulation does not penetrate the dermal layer of the skin through topical application, as illustrated in Figure 16. This observation underscores the safety of using the serum topically, as it does not reach the bloodstream by dermal penetration, which could potentially occur if it reached blood vessels.

Example 10: Comparison of viable bacteria cells of Lactobacillus contained in a commercial product with those contained in the serum of present invention on the skin.

The commercial product called "YUN ACN" repair face cream (Batch 4423- 752) from YUN Probiotherapy (Belgium) was purchased via internet and comprises Aqua, Glycerin, Cetyl Alcohol, Polyglyceryl-3 Rice Branate, Propylheptyl Caprylate, Butyrospermum Parkii Butter, Squalane, PEG-32, Caprylic/Capric Triglyceride, Cetearyl Alcohol, Prunus Amygdalus Dulcis Oil, Lactobacillus, Limnanthes Alba Seed Oil, Tocopheryl Acetate, Xanthan Gum, Allantoin, Algin, Sodium Benzoate, Potassium Sorbate, Hydrochloric Acid, Disodium EDTA, Tocopherol, Glycine Soja Oil, Citric Acid. The commercial products have been purchased, stored as recommended at the products until evaluation, and evaluated within the assigned product shelf life.

Commercial products with claimed viable Lactobacillus have been evaluated with respect to Colony Forming Units per mL on skin in a 2cm² x 2cm² area on the skin of healthy subject for a total time of 8h and compared to each baseline time 0 i.e. before application of commercial product.

The total viable cell count of Lactobacillus is determined as Colony Forming Units per mL product (CFU/mL) by spread plate method with MRS Agar pH 6.2 (Oxoid CM0361). 4 g product were mixed with 5 mL ddH20 using a vortex for mixing for 3 minutes at 550-700 strokes per minute. A serial dilution in ddHzO was prepared and plated on MRS Agar pH 6.2 plates incubated micro aerobically (5% CO2) at 37°C for 24- 48h. The colonies were counted, and results expressed as CFU/mL.

The data presented Figure 17 clearly indicate that serum according to the invention contains Lactobacillus at a concentration 400 times higher compared to that in the YUN cream. Furthermore, the population of lactobacillus demonstrates a remarkable increase of 51% within the serum according to the present invention after 8 hours of skin application. In contrast, the YUN cream shows a decrease in the bacterial population following the same duration of skin application. It is evident that the commercial product shows limited viability of Lactobacillus after 8hour application on skin of healthy subjects. Thus, it is evident that the commercial product YUN, claiming that to contain live Lactobacillus is highly unstable on skin. The L. plantarum concentration after reconstitution was at least 5 xlO⁶ CFU/mL and this is necessary for the bacteria to survive on the skin, data from healthy volunteers and from in vitro skin models show a viability (after 8h) which is superior to 50% of its starting values.

The concentration of specific probiotic in the serum of the present invention and the reasons for their sustained effectiveness are pivotal considerations. It is imperative to provide lucid instances elucidating the impact on both the skin and water subsequent to probiotic activation. Significantly, our product encompass a minimum threshold of Colony-Forming Units (CFU) >5xl0⁷ in water and 5xl0⁶ on skin. This stipulated quantity assures a perpetual availability on the skin's surface, with minor reductions becoming apparent only after a designated period (8 hours). This assertion finds validation in data obtained from biopsies and authentic skin samples.

Example 11: Determination of viable bacteria cells of Lactiplantibacillus plantarum during a 6-month storage stability study.

The study involved a 6-month storage stability analysis of the Lactobacillus plantarum inoculated from formulation (serum). The analysis was conducted at time points: TO, 2 weeks (T2), 1 month (T3), 3 months (T3), and 6 months (T6). Each sample was prepared according to the ISTISAN (Italian National Institute of Health Standards), which is a standardized method recommended for assessing viability and stability of microorganisms. The stability was assessed at temperature and relative humidity parameters of 25°C ± 2°C and 60 ± 5% RH and at temperature 5°C ± 3°C; data are respectively shown in Table 4 and Table 5, below.

Table 4: Storage stability results for microbial testing for contaminants and for L. plantarum at 25°C ± 2°C and 60 ± 5% RH.

Table 5: Storage stability results for microbial testing for contaminants and for L. plantarum at 5°C ± 3 °C. The results demonstrated the absence of the above microbial contaminant (yeast, moulds, 5. aureus, P. aeruginosa, C. albicans, E. coli in the formulation (serum)) after 6 month storage stability. Alive lactobacilli count in CFU/g was performed over the set-times and an average decrease of 50% resulted over a 6 months period at 25°C ± 2°C and 60 ± 5% RH when compared to initial TO. An average decrease of less than 50% resulted over a 6 month period when stored at 5°C ± 3 °C.

Bibliographic references

1. Navarro-Lopez V., Nunez-Delegido E., Ruzafa-Costas B., Sanchez-Pellicer P., Aguera- Santos J., Navarro-Moratalla L. Probiotics in the Therapeutic Arsenal of Dermatologists. Microorganisms,. 2021, 9, 1513.

2. Yu, Y., Dunaway, S., Champer, J., Kim, J., & Alikhan, A. Changing our microbiome: probiotics in dermatology. British Journal of Dermatology, 2020, 182, 39-46.

3. Zheng, J., Wittouck, S., Salvetti, E., Franz, C.M.A.P., Harris, H.M.B., Mattarelli, P., O'Toole, P.W., Pot, B., Vandamme, P., Walter, J., et al. A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. IntJ Syst Evol Microbiol 2020, 70, 2782-2858.

4. Gheziel, C.; Russo, P.; Arena, M.P.; Spano, G.; Ouzari, H.-L; Kheroua, O.; Saidi, D.; Fiocco, D.; Kaddouri, H.; Capozzi, V. Evaluating the Probiotic Potential of Lactobacillus plantarum Strains from Algerian Infant Feces: Towards the Design of Probiotic Starter Cultures Tailored for Developing Countries. Probiotics Antimicrob. Proteins 2019, 11, 113-123.

5. Seddik, H.A.; Bendali, F.; Gancel, F.; Fliss, I.; Spano, G.; Drider, D. Lactobacillus plantarum and Its Probiotic and Food Potentialities. Probiotics Antimicrob. Proteins 2017, 9, 111-122.

6. Peral, M.C., Huaman Martinez, M.A. and Valdez, J.C.. Bacteriotherapy with Lactobacillus plantarum in burns. International Wound Journal, 2009, 6, 73-81.

7. Arganaraz Aybar J.N., Ortiz Mayor S., Olea L., Garcia J.J., Nisoria S., Kolling Y., Melian C., Rachid M., Torres Dimani R., Werenitzky C., Lorca C., Salva S., Gobbato N., Villena J., Valdez J.C. (2022). Topical Administration of Lactiplantibacillus plantarum Accelerates the Healing of Chronic Diabetic Foot Ulcers through Modifications of Infection, Angiogenesis, Macrophage Phenotype and Neutrophil Response. Microorganisms, 2022, 10, 634.

8. Patent EP 2364712 Bl published on March 6, 2013.

9. International application WO 2009/133519 A2 published on November 5, 2009. 10. Patent application FR 2 913 337 Al published on September 2008.

11. International application WO 2020/127637 Al published on June 25, 2020.

12. Patent application CN 112226429 A published on January 15, 2021.

13. International application WO 2020/007906 Al published on January 9, 2020. 14. Nakai, H., Hirose, Y., Murosaki, S., Yoshikai, Y. Lactobacillus plantarum L-137 upregulates hyaluronic acid production in epidermal cells and fibroblasts in mice. Microbiol and Immunol. 2019, 63, 367-378.

15. Kober, M.M., Bowe W.P. The effect of probiotics on immune regulation, acne, and photoaging. IntJ Womens Dermatol. 2015, 1, 85-89.

Claims

1. A two-phase formulation comprising:

- a primary powder phase comprising at least one still alive strain of probiotic bacteria in a freeze-dried state and at least one gelifying agent, and

- a complementary aqueous and sterile phase, physically separated from the primary powder phase.

2. The two-phase formulation according to claim 1, wherein said at least one strain of probiotic bacteria belongs to the Lactiplantibacilli genus and, in particular is Lactiplantibacillus plantarum.

3. The two-phase formulation according to claim 1 or 2, wherein said at least one gelifying agent is hyaluronic acid.

4. The two-phase formulation according to any one of claims 1 to 3, wherein said primary powder phase further comprises at least one sugar and, the latter being preferably mannitol, sorbitol or a mixture thereof.

5. The two-phase formulation according to any one of claims 1 to 4, wherein said primary powder phase and/or said complementary aqueous and sterile phase may further comprise at least one vitamin, the latter being preferably vitamin B-complex or a salt thereof.

6. A device for the preparation of an active probiotic composition, comprising:

- a first container containing the primary powder phase as defined in any one of claims 1 to 5, and

- a second container containing the complementary aqueous and sterile phase as defined in claim 1 or 5, the first container and the second container being configured to be connected to each other and to allow mixing of said two phases to prepare an active probiotic composition.

7. The device according to claim 6, wherein at least one of said first container and said second container is a syringe (1, 10).

8. The device according to claim 6 or 7, wherein at least one of said first container and said second container is a vial (2), a pouch (100, 110) or a capsule and in particular a glass vial.

9. The device according to any one of claims 6 to 8, further comprising a connector (3, 30) configured to connect and seal said first container with said second container.

10. A method for preparing an active probiotic composition comprising the steps of :

- connecting together the first container and the second container of the device as defined in any one of claims 6 to 9, and

- adding the complementary aqueous and sterile phase as defined in claim 1 or 5 contained in said second container to the primary powder phase as defined in any one of claims 1 to 5 contained in said first container and mixing together both phases, whereby an active probiotic composition is prepared.

11. The method according to claim 10, wherein the mixing is performed by applying pressure up and down and/or by shaking.

12. An active probiotic composition prepared by the method according to claim 10 or 11, wherein said active probiotic composition is an aqueous suspension consisting of at least one probiotic bacteria strain, at least one gelifying agent, at least one sugar as previously defined, at least one vitamin and water.

13. The active probiotic composition according to claim 12, wherein said active probiotic composition comprises at least one probiotic bacteria strain in an amount comprised between 10⁴ alive cells and 10¹² alive cells per mL of said composition.

14. A kit comprising a device as defined in any one of claims 6 to 9, and a repository to hold the active probiotic composition according to claim 12 or 13, and ready to immediately apply on the skin.

15. The active probiotic composition according to claim 12 or 13 for use in preventing and/or treating an epidermis pathology selected in the group consisting of acne, eczema, atopic dermatitis, alopecia, dysbiosis caused Cutibacterium acne, skin infections caused by Candida auris, vaginal mucosal layer prone to Candida infection and/or by Staphylococcus aureusfor a subject in need, wherein the active probiotic composition is topically applied on the skin, the scalp or a mucosa of said subject.

16. Cosmetic and non therapeutic use of the active probiotic composition according to claim 12 or 13 for modifying the top skin microbiome and/or for restoring the balance in skin microbial communities and/or restoring an healthy skin microbial communities and/or for restoring skin appearance and/or for improving the aesthetic appearance of damaged area of the skin in a subject, wherein the active probiotic composition is topically applied on the skin, the scalp or a mucosa of said subject.