RU2794128C2 - Bacterial secret for use in treatment of skin damages - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention concerns the use of the bacterial secretome of the bacterium deposited at CNCM under the number I-4290 in the treatment of skin lesions. The said secret is produced by a method comprising cultivating the said bacterium in a culture medium and under suitable conditions for its growth, liquid/solid separation, and isolating a liquid phase, wherein said liquid phase contains secret. A basic buffer chosen from Tris buffer, arginine buffer and mixtures thereof is added to the resulting liquid phase, the resulting buffered liquid phase is incubated for 1-7 hours at a temperature of 1 to 6° With obtaining the specified secretome. Also provided is a dermatological composition for use in the treatment of skin lesions, containing an effective amount of said bacterial secretome and a dermatological excipient suitable for topical application.

EFFECT: invention is effective in the treatment of skin lesions.

10 cl, 4 dwg, 2 tbl, 5 ex

Description

FIELD OF THE INVENTION

The present invention relates to a new use of bacterial secretome in the field of treatment of skin lesions and, more specifically, wound healing. This invention also relates to cosmetic or dermatological compositions containing such a bacterial secretion as an active agent.

PRIOR ART

Wound healing is a complex and dynamic biological process that involves the interaction of many local and systemic factors in the repair of normal tissue. Healing progresses in three interdependent phases: hemostasis and inflammation, proliferation and remodeling (General principles of wound healing. Witte MB, Barbul A. Surg Clin North Am. 1997 June; 77(3):509-28.). Proliferation involves three clearly observable processes: granulation, contraction, and re-epithelialization.

During granulation, it is observed that the cells involved in the repair process proliferate and migrate to the wound bed. For example, there are macrophages, fibroblasts and endothelial cells. Macrophages constantly release chemotactic factors and growth factors. Fibroblasts build the new cell matrix needed for cell growth at the base of the wound. This scaffold formation supports cell migration. Finally, endothelial cells trigger the formation of vascular buds that will establish new capillaries, thereby restoring perfusion and supplying oxygen and nutrients important for the metabolic activity of cells in the wound.

Wound contraction is the mechanism for reducing wound size, and fibroblasts play a leading role in this contraction.

Re-epithelialization consists in the regeneration of the epidermis that covers the wound with the formation of an effective barrier against the external environment, capable of becoming pigmented and restoring its sensory and immune functions. It thus involves the cellular processes of migration and proliferation of keratinocytes, but also the differentiation of this new epithelium and the restoration of the basement membrane that connects the dermis and epidermis. When the migration of basal cells towards the center of the wound ensures that the edges of the wound meet, a wave of cellular mitosis occurs to fill in the spaces left by the migration and to provide cells for the epithelial tissue in a three-dimensional regeneration.

Proliferation stages of keratinocytes, fibroblasts and endothelial cells can be considered as one of the functional phenomena demonstrating the healing activity of the active agent. Increased fibroblast proliferation would be involved in deep wound healing (reaching the dermis), while increased keratinocyte proliferation would be involved in re-epithelialization.

Healing also imposes cosmetic problems with healing defects or poor healing, causing unsightly and mostly permanent visible marks such as thick scars or keloids.

All cosmetic and dermatological surgical procedures, as well as pathological and non-medical traumatic injuries (scratches, cuts, scrapes, burns) leave scars. One of the main skills of plastic surgeons is scar control.

For example, pressure has been used against burn scars for many years and has recently been combined with a silicone sheet applied directly to the scar.

There remains a need to provide new compositions to promote healing of damaged skin and to improve the cosmetic appearance of scars.

For the first time and unexpectedly, the Applicant has shown beneficial properties in tissue regeneration and in the healing of skin lesions of a bacterial secretome derived from the bacterial strain (or bacterium) LMB64 isolated from ground water.

This bacterium has been described by the applicant in patent application WO 2012/085182. More specifically, this bacterium has been described as such for its anti-inflammatory activities. Even more specifically, extracts named S0, E0 and ES0, consisting of the culture supernatant separated from the biomass - lysed cell biomass, and the supernatant after culture incubation at basic pH for several hours, respectively, have been described and exemplified. Only the pharmacological activities of E0 and ES0 extracts were analyzed and these E0 and ES0 extracts were shown to be able to induce cytokines and Langerhans cell maturation (for E0) as well as activation of TLR2 (Toll-like receptor 2)/TLR4/TLR5, PAR antagonists , and induction of antimicrobial peptides (for ES0). These results suggested the use of such extracts in the treatment of inflammatory diseases such as pruritus, psoriasis, eczema or atopic dermatitis.

Contrary to all expectations, the applicant shows in this document that the secretion, which does not consist of the same components as the above extracts ES0 and E0, but mainly consists of biomolecules secreted and excreted into the culture supernatant by said LMB64 bacterium, has unexpected healing properties.

DETAILED DESCRIPTION OF THE INVENTION

According to the first embodiment (1), the present invention relates to a bacterial secretion of a non-pathogenic gram-negative bacterium belonging to the class Betaproteobacteria, subfamily Neisseriaceae, containing 16S rRNA containing the sequence of SEQ ID NO: 1 or any sequence having at least 80% identity with the sequence SEQ ID NO: 1, for use in the treatment of skin lesions,

moreover, the specified secret is likely to be obtained or obtained by a method including the following stages:

- a) cultivating said bacterium in a culture medium and under suitable conditions for its growth,

b) liquid/solid separation and separation of the liquid phase,

- c) obtaining said secretome containing biomolecules secreted by said bacterium into the liquid phase.

According to a second embodiment of the present invention, the bacterial secretion for use in the treatment of skin lesions according to the present invention is characterized in that a basic buffer is added to the liquid phase obtained in step b), and that the resulting buffered liquid phase is optionally left in contact for 1-7 hours preferably at a temperature of from 1 to 6°C.

The bacterium LMB64 has been characterized and identified as belonging to the class Betaproteobacteria, subfamily Neisseriaceae, and possibly a new, as yet unidentified genus. Sequence analysis of the gene encoding 16S ribosomal RNA (rRNA) made it possible to place this bacterium close to the genera Chromobacterium, Paludimonas, Lutelia, and Glubenkiana, with which it shares 95% sequence similarity.

This non-pathogenic bacterium is Gram-negative and has been isolated from groundwater.

More specifically, the LMB64 bacterium is rod-shaped with a length of roughly 2.3 µm (plus/minus 0.3) and a width of roughly 1.0 µm (plus/minus 0.1). A distinctive feature of this bacterium is the presence of a polar flagellum.

The gene encoding the 16S rRNA was almost completely sequenced (1487 bp, corresponding to the sequence of SEQ ID NO: 1). The LMB64 bacterium has a 10948 bp circular plasmid. This plasmid has been fully sequenced and the sequence is shown in SEQ ID NO: 2.

According to another embodiment, the bacterium from which the secretion of the invention originates contains at least one plasmid containing the sequence of SEQ ID NO: 2, or any sequence having at least 80% identity with the sequence of SEQ ID NO: 2, mainly on at least 85%, at least 90%, at least 95%, even at least 97%, and even more preferably at least 98% identity with SEQ ID NO: 2.

In addition, the bacterium from which the secretion originates is a non-pathogenic Gram-negative bacterium belonging to the class Betaproteobacteria, subfamily Neisseriaceae, said bacterium containing a 16S rRNA containing the sequence of SEQ ID NO: 1 or any sequence having at least 80% , even at least 90%, at least 95%, at least 97% identity with the sequence of SEQ ID NO: 1, and the specified bacterium contains at least one plasmid containing the sequence of SEQ ID NO: 2, or any sequence having at least 80% identity with the sequence of SEQ ID NO: 2, preferably at least 85%, at least 90%, at least 95%, even at least 97% and even more preferably at least 98% identity with the sequence of SEQ ID NO: 2.

An example of such a bacterium is the LMB64 strain, which was deposited on behalf of the Applicant with the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, Paris, April 8, 2010 under the number I-4290.

With this genotypic information, combined with the characteristics regarding growth in a sulfur-free environment and the non-filamentous nature of this bacterium, one skilled in the art would have no difficulty in finding/identifying another bacterium, providing a secretome of the invention. Such an identification of another bacterium, naturally genotypically slightly different, but having all the phenotypic criteria of the invention in terms of secretome, can be obtained after selection research, which is by no means conclusive, and this is based on the information contained in this application, and which is contained in WO 2012/085182, combined with the general knowledge of a person skilled in the art.

In the context of the present invention, "percent identity" between two nucleic acid sequences refers to the percentage of identical nucleotides between two sequences to be compared, obtained after the best alignment (optimal alignment), this percentage being purely statistical, and the differences between the two sequences are distributed along their entire length randomly. Comparison of sequences between two nucleic acid sequences is usually done by comparing these sequences after they have been optimally aligned, where said comparison can be done per cut or "comparison window". Optimal alignment of sequences for comparison can be performed, in addition to manual comparison, using the local homology algorithm of Smith and Waterman (1981) [Ad. app. Math. 2:482], through the local homology algorithm of Needleman and Wunsch (1970) [J. Mol. Biol. 48:443], through the Pearson and Lipman (1988) similarity search method [Proc. Natl. Acad. sci. USA 85:2444] or by a computer program using these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics, Genetics Computer Group, 575 Science Dr., Madison, WI, or BLAST N or BLAST P comparison program) .

The percentage identity between two nucleic acid sequences is determined by comparing these two optimally aligned sequences, where the nucleic acid sequence to be compared may include insertions or deletions compared to a control sequence for optimal alignment between the two sequences. Percent identity is calculated by determining the number of positions for which a nucleotide is identical between two sequences, dividing this number of identical positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage identity between the two given sequences.

For example, one can use the BLAST program - "BLAST 2 sequences" (Tatusova et al., "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250) available at http: //www.ncbi.nlm.nih.gov/gorf/bl2.html, with default parameters (in particular, in relation to the parameters "penalty for creating a gap": 5 and "penalty for lengthening a gap": 2; moreover, with the selected matrix is, for example, the matrix "BLOSUM 62" proposed by the program). The percentage identity between the two sequences to be compared is calculated directly by the program. Other programs such as "ALIGN" or "Megalign" (DNASTAR) can also be used.

The bacterium of the invention includes the bacterium LMB64, which contains at least one plasmid containing the sequence of SEQ ID NO: 2, or any sequence having at least 80%, preferably 85%, 90%, 95% identical or 98% identity with the specified sequence of SEQ ID NO: 2. Other characteristics of the specified bacterium LMB64 will be described in detail below in the examples.

In addition, the bacterium LMB64 was deposited on behalf of the applicant with the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, Paris, on April 8, 2010 under the number I-4290.

According to another embodiment, the present invention also relates to a bacterial secretion for use in the treatment of skin lesions according to the second embodiment, characterized in that the main buffer is selected from Tris buffer, arginine buffer and mixtures thereof.

In another embodiment, the present invention relates to a bacterial secretion for use in the treatment of skin lesions according to one of the preceding embodiments, characterized in that step b) after liquid/solid separation is followed by step b') of clarifying the liquid phase or buffered liquid phase by filtration before stage c).

In another embodiment, this invention relates to a bacterial secretion for use in the treatment of skin lesions according to the previous embodiment, characterized in that the filtration is carried out with a cut-off of 0.2 μm.

According to another embodiment, the invention also relates to a bacterial secretion for use in the treatment of skin lesions according to one of the preceding embodiments, characterized in that said biomolecules consist of peptides, proteins and secondary metabolites secreted by said bacterium.

Another embodiment of the present invention also provides a bacterial secretome for use in the treatment of skin lesions according to one of the previous embodiments, characterized in that the bacterium contains at least one plasmid containing the sequence of SEQ ID NO: 2 or any sequence having at least 80% -th identity c SEQ ID NO: 2.

According to another embodiment, this invention also relates to a bacterial secretion for use in the treatment of skin lesions according to one of the previous embodiments, characterized in that the bacterium is a bacterium deposited in CNCM on April 8, 2010 under the number I-4290.

In general, the term secretome is used to describe all biomolecules secreted and released into a culture medium and solubilized in that medium by a cell, tissue, or organism. In the present case, it is to be understood that the term "secretion" refers to all biomolecules secreted and excreted by the bacterium LMB64, and this occurs without modification, damage or lysis of the bacterial cell. More specifically, these biomolecules are mainly composed of proteins, peptides and secondary metabolites secreted and excreted by the bacterium into the culture medium.

Bacteria reproduce by dividing in two, i.e. each bacterium grows and then divides into two daughter cells separated by a separating septum formed by the cell wall. During division, DNA and other components are duplicated. Cell division involves different enzymatic systems of synthesis and degradation.

Bacterial growth is an orderly increase in all components of a bacterium. It leads to an increase in the number of bacteria. During growth, on the one hand, there is a depletion of the culture medium in nutrients and, on the other hand, an enrichment in biomolecules secreted and released into the culture medium by the bacterium and solubilized in this medium, as well as by-products of metabolism.

It should be understood that the expression "culture medium" or "medium" refers to any medium containing at least the necessary nutrients for bacterial growth and reproduction. Bacteria can be grown in liquid, solid or semi-solid media. Preferably, the culture medium is a liquid medium capable of recovering the culture supernatant containing the secretion.

A suitable culture medium contains nutrients that stimulate bacterial growth and reproduction. In general, a suitable culture medium will contain water, a carbon source, a nitrogen source, and salt. By way of illustration, a specific exemplary culture medium is described in the examples below.

It is to be understood that the expression "secondary metabolites" refers to small molecules that are produced, secreted and released into the culture medium by the bacterium of the invention, in particular the bacterium LMB64. By way of illustration, such secondary metabolites may be soluble molecules such as bacteriocins, nonribosomal peptides, siderophores, lipopeptides, or polyketides, or molecules such as terpenes, pyrazines, indoles, or sulfide derivatives. The proteins present in the secretome range in size from 10 kD to 100 kD, preferably from 18 kD to 98 kD, and preferably the predominant protein, which is roughly 38 kD.

In practice, the secretion of a bacterium of the invention, for example, and in particular the bacterium LMB64, can be obtained from a culture in a culture medium that allows the growth, development and reproduction of a bacterium (for example, a bacterium described below), simply by isolating the culture supernatant containing the secreted biomolecules constituting the secret of the invention. In specific terms, due to the fact that these biomolecules are secreted and excreted by the bacterium and solubilized in the medium, there is no need to carry out the step of treating said bacteria (mechanical or chemical, inducing complete or partial cell lysis, or release of proteins or components of the wall, membrane or periplasmic spaces). Culture supernatant components (hereinafter secret) can be isolated from insoluble solid cell components by any liquid/solid separation techniques. More specifically, by means of techniques known to those skilled in the art, it is therefore possible to isolate a liquid fraction containing solubilized biomolecules from a solid fraction containing primarily whole cells and cell debris containing surface proteins and/or proteins localized in the periplasmic space. bacteria.

By way of illustration, liquid/solid separation can be accomplished by a technique selected from, for example, centrifugation, settling, filtration, ultrafiltration, decantation, drying.

Preferably, the liquid/solid separation is carried out by centrifugation of the bacterial culture in order to separate the solid phase, i. sediment containing cells and cell debris; and the liquid phase, i.e. supernatant containing soluble molecules, i.e. secret.

Supernatant, i.e. the liquid phase can be used as such or after simple filtration in order to remove possibly present cell debris and to sterilize it. Concentration may also be considered, for example by diafiltration. Secretion proteins can also be isolated by ammonium sulfate precipitation.

As indicated above, the method for producing a bacterial secretome according to the invention is characterized in that a basic buffer is added to the liquid phase obtained in step b), and that the resulting buffered liquid phase is optionally left in contact with such a buffer for 1-7 hours, in particular , at low temperature, preferably at a temperature of from 1 to 6°C.

The addition of a basic buffer to the supernatant improves the solubility and/or stability of proteins and peptides in solution.

Preferably, this basic buffer is a Tris buffer or an arginine buffer, or a Tris-arginine mixture. Preferably it is a Tris-arginine buffer.

The amount of basic buffer, preferably Tris-arginine, added to the liquid phase is such that the resulting buffered liquid phase has an arginine concentration of 100 mM to 500 mM and/or a Tris concentration of 10 mM to 90 mM.

The pH of the buffered liquid phase may be from 8 to 12 and preferably from 9 to 11.

The use of the main buffer (Tris, arginine or Tris-arginine) ensures the stabilization of proteins in solution and avoid their aggregation over a long period of storage.

A preferred method for cultivating the bacterium of the invention, in particular the LMB64 strain, may include three steps.

• The first inoculum can be prepared in an Erlenmeyer flask in a suitable medium containing minerals, proteins and carbohydrates appropriate for the preculture.

• A second pre-fermentation culture is then prepared batchwise in a second medium similar or identical to the first medium (or any equivalent medium).

• Finally, a fed-batch fermentation is carried out in a medium identical or similar to the second medium to obtain a high cell density.

The centrifugation step then removes the cells and cell debris and recovers the culture supernatant.

This buffered supernatant or buffered liquid phase can then be clarified by filtration.

As shown above, step b) of liquid/solid separation is followed by step b') of clarifying the liquid phase or buffered liquid phase, for example by filtration, before step c).

Filtration can be carried out by any suitable means providing clarification of the liquid phase, or, if applicable, the buffered liquid phase. Such clarification by filtration removes suspended particles not removed during the liquid/solid separation step and aims to produce a clear secretome.

The filtration can be carried out by any of the filtration, ultrafiltration or diafiltration methods.

Preferably, the filtration is carried out by filtration through a filter or cartridge filter having a cut-off of 0.4 µm, preferably 0.2 µm. In this case, the bacterial secretion is characterized in that the secreted peptides, proteins and secondary metabolites are 0.2 μm or less in size.

Preferably, electrostatically uncharged filters or pre-filters can be used in order to avoid any absorption of the biomolecules responsible for all or part of the activity.

The exact nature of the environment and the different stages will be described in greater detail in the Examples. It is to be understood that any modification of the method, medium, or sequences of steps that would be apparent to a person skilled in the art with respect to the present disclosure should be considered to be within the scope of this patent application.

As mentioned above, the invention also relates, in another embodiment, to a bacterial secretome for use in the treatment of skin lesions according to one of the preceding embodiments, characterized in that these skin lesions are selected from the group consisting of abrasions, burns, sunburn, scratches, cuts , scrapings, stitches.

This invention also relates to bacterial secretion for use in the treatment of skin lesions according to one of the previous embodiments for use in alleviating healing defects.

The invention also relates, in another embodiment, to a bacterial secretome for use in the treatment of skin lesions according to one of the preceding embodiments, characterized in that these skin lesions occur after trauma, surgery, a dermatological procedure, or a cosmetic medical procedure.

Advantageously, the invention also relates to a bacterial secretion for use in the treatment of skin lesions according to one of the preceding embodiments, characterized in that it is combined with a copper salt or a zinc salt.

In another embodiment, the invention also relates to a bacterial secretion for use in the treatment of skin lesions according to one of the previous embodiments, characterized in that the treatment of skin lesions is to improve the healing and repair of the skin.

This invention further relates, in another embodiment, to a cosmetic treatment method for alleviating skin healing defects, comprising applying an effective amount of a composition containing an effective amount of a bacterial secretome as defined in one of the preceding embodiments, in combination with a cosmetically acceptable excipient, and formulated in a suitable form for local application.

Advantageously, the invention also relates to a bacterial secretion for use in the treatment of healing defects according to one of the preceding embodiments, characterized in that it is combined with a copper salt and/or a zinc salt.

Therefore, the present invention further relates in another embodiment to the use of a bacterial secretome as defined in one of the preceding embodiments, to mitigate the cosmetic treatment of skin healing defects. According to a specific embodiment, the secret of the invention is used in combination with a copper salt and/or a zinc salt.

In another embodiment, this invention relates to a dermatological composition comprising a bacterial secretion as defined in one of the previous embodiments and a suitable dermatological excipient for topical application. In a preferred embodiment, the dermatological composition further comprises a zinc salt and/or a copper salt.

In another embodiment, this invention further relates to a dermatological composition according to one of the preceding embodiments for use in the treatment of skin lesions. In a preferred embodiment, the dermatological composition further comprises a zinc salt and/or a copper salt.

In another embodiment, the present invention relates to a cosmetic composition comprising a bacterial secretion as defined in one of the preceding embodiments and a suitable topical cosmetic excipient. This cosmetic composition may further include a zinc salt and/or a copper salt.

Finally, in another embodiment, the present invention relates to the use of a cosmetic composition according to the previous embodiment for alleviating cosmetic treatment of skin healing defects.

According to one embodiment, this invention relates to a bacterial secretion, as defined in one of the previous embodiments, for topical use to stimulate fibroblast proliferation.

According to one embodiment, this invention relates to a bacterial secretion, as defined in one of the previous embodiments, for topical application to stimulate the proliferation and/or migration of keratinocytes.

According to one embodiment, this invention relates to a bacterial secretion, as defined in one of the previous embodiments, for topical use in the treatment of skin lesions.

According to one embodiment, this invention relates to bacterial secretion, as defined in one of the previous embodiments, for topical use to improve healing and repair of the skin.

This invention also relates to a composition for accelerating skin repair in order to restore the integrity and quality of the skin, containing as an active principle secret, as defined in one of the previous embodiments. More preferably, the composition may further comprise a copper salt and/or a zinc salt.

Preferably the concentration of secretome in the composition according to the invention is from 0.05% to 10%. More preferably it is from 0.1% to 5%.

The composition of the invention further comprises one or more conventional dermatologically and/or cosmetically compatible excipients known to the person skilled in the art, in order to provide a composition for topical application, such as emulsifiers, thickeners, gelling agents, water-binding agents, spreaders, stabilizers , dyes, fragrances and preservatives.

As used herein, "dermatologically or cosmetically acceptable" is intended to mean one that is useful in the preparation of a dermatological or cosmetic composition, that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and that is dermatologically or cosmetically acceptable. applications, and in particular dermatological or dermo-cosmetic applications, in particular by topical application.

The compositions of the invention are advantageously intended for topical application, in particular to the skin.

The composition according to the invention may be in the form of an emulsion, dispersion, or aqueous or oily lotion.

In another specific embodiment, the dermatological or cosmetic composition of the invention comprises at least one other active, more specifically at least one moisturizing active.

This other active principle can thus in particular be selected from the group consisting of the following:

• Well-known healing or restorative agents: panthenol, madecassoside, allantoin, aloe vera, honey;

• Anti-infective agents: trace elements such as copper-zinc, honey

• Moisturizers and nourishers: glycerin, shea butter, natural waxes, hyaluronic acid, fatty acids

• Calming agents: allantoin, bisabolol, panthenol, enoxolone

• UV filters (ultraviolet radiation).

Preferably, the second active principle will be a trace element of the copper-zinc type. In this preferred embodiment, the composition according to the invention will thus include secretion and a micronutrient such as copper-zinc.

As mentioned, the dermatological or cosmetic composition of the invention containing secretion may include a copper salt or a zinc salt. Preferably the copper salt will be copper sulfate. Advantageously, the zinc salt will be selected from zinc oxide and zinc sulfate.

The amount of copper sulfate in the composition according to the invention may vary from 0.05 to 0.5% of the mass. compositions, preferably from 0.1 to 0.25% of the mass. compositions, more preferably roughly 0.2% of the mass. compositions.

The amount of zinc sulfate in the composition according to the invention may vary from 0.05 to 0.5% of the mass. compositions, preferably from 0.1 to 0.4% of the mass. compositions, more preferably from 0.1 to 0.2% of the mass. compositions.

The amount of zinc oxide in the composition according to the invention may vary from 0.5 to 10% of the mass. compositions, preferably from 1 to 5% of the mass. compositions, more specifically roughly from 2 to 4% of the mass. compositions.

In one embodiment of the present invention, a composition according to any of the preceding embodiments is contemplated, characterized in that it further comprises at least one other active principle selected from healing agents, anti-infective agents, humectants, soothing agents, and mixtures thereof. According to another specific embodiment, at least one other active principle will be selected from humectants, healing agents, soothing agents and mixtures thereof, preferably humectants. Such an active principle will preferably be used in dermatological compositions.

The present invention also relates to a method for treating and healing skin lesions, comprising administering, preferably locally, an effective amount of a composition containing a secret according to the invention as the active principle.

The compositions of the invention are particularly intended for the care of stubbornly damaged skin.

Skin lesions can appear after trauma or medical or surgical procedures, whether they are invasive or non-invasive, curative or for cosmetic purposes.

Invasive medical or surgical procedures include, for example: surgical procedures (excision, shaving) with or without sutures, cryotherapy, laser ablation, moderate to severe chemical peels, sutures, mesotherapy or curettage.

Post-traumatic skin injuries, for example, after a slight external injury to the skin, include, for example, cuts, scrapes, scrapes and superficial burns or sunburn.

Superficial non-invasive medical procedures requiring a healing product that accelerates skin repair and is suitable for long-term use (until the skin is completely healed) include, for example, light chemical peels, laser hair removal, and superficial vascular (rosacea) treatments.

The treatment of skin and mucosal lesions according to the invention may particularly include the treatment of cuts, stitches, abrasions, scrapings, scratches, scars following a surgical or cosmetic dermatological procedure, superficial burns, sunburn.

The present invention further relates to the use of the cosmetic composition of the invention for improving the healing and repair of the skin, and in particular for alleviating healing defects.

The invention will be better understood by reading the following examples, which illustrate the invention without limiting its scope.

These examples refer to the following FIGS.

FIG. 1: SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel of secretome proteins.

Lane 1 (M - molecular weight markers, azure).

Lanes 2-5: (Sec - secret before adding arginine buffer) - load 10 µl, 15 µl, 20 µl and 30 µl, respectively.

Lanes 6-9: (Sec + Arg - secreted after addition of arginine buffer) - loading 10 µl, 15 µl, 20 µl and 30 µl, respectively.

FIG. 2: % stimulation of proliferation (mean plus/minus SEM (standard error of the mean)) of normal human fibroblasts treated with different products compared to control cells (n is 3 donor groups).

FIG. 3: % stimulation (mean plus/minus SEM) of migration of a HaCaT keratinocyte cell line treated with different compounds compared to control cells (n is 2).

FIG. 4: Wound re-epithelialization (mean plus/minus SEM) after 48 hours of application to pig ear explants of different products compared to repair cream control (n = 10 independent donors).

Example 1: culture of the bacterium LMB64

As a non-limiting example, the preferred culture medium contains ammonium chloride, magnesium sulfate and yeast extract. It should also be noted that, as follows from WO 2012/085182, among others, other similar media can be used and are thus to be considered an integral part of the present description. Any adaptation by those skilled in the art should also be considered part of the present invention.

A typical culture method is described below. It should be recalled here that this example is for illustrative purposes only and should in no way be construed as limiting.

The LMB64 strain is grown in three stages, namely: initial inoculation, pre-cultivation (or pre-fermentation) in batch mode, and finally cultivation, but in feed mode (addition of glucose).

Inoculum: To inoculate an Erlenmeyer flask containing 1000 ml of sterile medium, use a WCB (Working Cell Bank) LMB64 tube. This Erlenmeyer flask is then placed in an incubator shaker and shaken. When a sufficient cell density of the broth is reached, the culture is stopped. The cells are then cooled before being transferred to the pre-fermenter.

Pre-cultivation: The pre-fermenter is then filled roughly with 16 liters of medium and then completely sterilized.

Two satellite flasks are connected to the pre-fermenter after the vessel has been sterilized and then to the following additional blocks:

• a flask containing a sterile 50% glucose solution. This solution (intermittent pre-cultivation with glucose) is immediately transferred to the culture medium to achieve an initial glucose concentration of 20 g/l.

• An Erlenmeyer flask containing the inoculum described above in the Inoculum step is inoculated in the pre-fermenter.

Start precultivation and then adjust automatically. As an example, the following parameters can be mentioned: temperature, shaking speed, pressure, air flow rate or PO ₂ .

Cell growth is monitored by measuring optical density at 620 nm. Pre-cultivation is stopped by cooling when it reaches sufficient density.

Cultivation: The fermenter is then filled with 127 liters of medium adjusted to pH 7.0 and then completely sterilized. Three satellite flasks are used as follows:

• a flask containing a sterile glucose solution. This solution is immediately transferred to the culture medium to achieve an initial glucose concentration of 20 g/l.

• flask containing defoamer. This defoamer will be automatically added during cultivation to control the level of foam in the tank.

• flask with glucose for feeding. This solution will be added throughout the culture to stimulate cell growth.

The culture is started and then adjusted automatically. As an example, the following parameters can also be mentioned: temperature, pH, agitation, pressure, air flow rate, PO ₂ .

After the glucose initially present in the medium is depleted (increased PO ₂ level), the addition of glucose make-up solution is started, thus ensuring cell growth at high density. Fermentation is stopped after complete consumption of glucose. At this stage, the fermentation should automatically cool down. Throughout the cultivation, cell growth is monitored by measuring the optical density at 620 nm. The amount of dry biomass (g/l) obtained at the end of cultivation is determined using a weighing method.

Example 2: secretome extraction

The example below is given as an illustration of the preferred embodiment, but should not be construed as limiting.

The secret is generally obtained after centrifuging the result of the culture step in order to remove cells, surface proteins and proteins located in the periplasmic space of the bacterium. This centrifugation step is followed by the addition of Tris-arginine basic buffer to the supernatant. A last step of filtering the supernatant is also possible. Any adaptation by those skilled in the art should also be considered part of the present invention.

Centrifugation: The transfer line from the fermenter to the centrifuge is sterilized. The fermentation wort is then separated by continuous centrifugation in a centrifuge. Centrifugation is performed at a speed of 150 l/h (plus/minus 30 l/h) with a vessel rotation speed of 10900 plus/minus 1000 rpm. The supernatant is withdrawn into a container equipped with a disposable bag. The weight of the supernatant is measured on the weighing platform.

Addition of arginine: Tris-arginine extraction buffer is sterilized and then transferred to a disposable bag containing the culture supernatant. Required contact time from 1 to 7 hours. The target concentration of Tris and arginine after addition to the disposable bag is roughly 0.3 M L-arginine and 20 mM Tris.

The total volume after the addition of extraction buffer is roughly 240 L.

Filtration: two successive filtration steps are carried out in order to clarify the supernatant and obtain a clear, germ-free secretion. The filtration is controlled by the filtration/distribution system. A disposable depth filter cartridge is placed in its filter housing. All filtered product is sterilely isolated in a container equipped with a disposable bag. The filtered product bag is weighed on the weighing platform and then stored at +5°C until distribution.

Gel electrophoresis of secretome proteins: see FIG. 1.

Used Bis-Tris SDS-PAGE in gel (4-12%) under denaturing and reducing conditions, Coomassie blue detection of loadings of different volumes of culture supernatants (10, 15, 20, 30 µl, respectively) before and after the addition of arginine buffer. In both cases, different protein bands are observed ranging in size from 14 to 100 kDa, including a main band of roughly 38 kDa.

Example 3: Effect of Secretome on Proliferation of Normal Human Fibroblasts

The technique used is that of incorporating the nucleotide, 5-bromo-2'-deoxyuridine (BrdU), a thymidine analogue, into the DNA of S-phase cells at 37°C. This technique provides a quantitative measurement of cells whose progression in the cell cycle is characteristic of a proliferating cell (phase S or DNA synthesis).

Fibroblasts are seeded in a 96-well plate and then incubated for 72 h in the presence of compounds to be analyzed, EGF (epidermal growth factor) as a positive control and 0.4; 0.6 and 1% secretome. BrdU incorporation occurs within the last 24 hours and is quantified using the BrdU ELISA Kit (Product No. 11647229001, Roche Diagnostics).

% of control is calculated according to the following formula:

(value / mean of control) × 100

Stimulation in % is calculated according to the following formula:

Mean Stimulation in % = (Mean (Value / Mean of Control) × 100) - 100

SEM = standard deviation / √n

Statistical analyzes are performed using Student's t-test. These studies were carried out on 3 groups of donors.

The positive control - EGF - strongly, significantly and reproducibly stimulated fibroblast proliferation in 3 donor groups (95% stimulation). This confirms the experiments. These results indicate that secretion assayed at 0.4%, 0.6% and 1% significantly stimulated the proliferation of normal human fibroblasts. Table 1 below summarizes the results of BrdU incorporation (mean plus/minus SEM) with normal human fibroblasts (by ELISA) after 72 hours of incubation in the presence of the tested compounds (n is 3 donor groups). The results are shown in FIG. 2.

Example 4: Effect of Secretome on the Migration of the HaCaT Keratinocyte Cell Line

These studies were performed using the Oris cell migration assay (Platypus Technologies) according to the standard protocol recommended by the manufacturer. Preferably, HaCaT cells (human keratinocyte cell line) are seeded in a 96-well plate. The Oris™ plugs are removed and the compounds to be evaluated are added to the culture medium. Incubation continues for 24 hours to ensure cell migration. A cell-permeable fluorescent label is then added to allow visualization of the cells in the zone delimited by the plugs.

Cell migration is assessed by photographing the wells and measuring the migration surface area in mm ² .

% control is calculated according to the following formula:

(value / mean of control) × 100

Stimulation in % is calculated according to the following formula:

Mean Stimulation in % = (Mean (Value / Mean of Control) × 100) - 100

SEM = standard deviation / √n

Statistical analyzes are performed using Student's t-test.

These studies are carried out on 6 technical repetitions and n equals 2 independent experiments.

Positive controls - TGF-b and fetal calf serum (FCS) - strongly, significantly and reproducibly stimulated cell migration (51% and 176% stimulation, respectively). This confirms the experiments. The results show that secretion assayed at 0.4%, 0.6% and 1% significantly stimulated HaCaT keratinocyte migration. Table 2 below summarizes the results of HaCaT cell migration after 24 hours of incubation with different compounds (n is 2). These results are shown in Fig. 3.

Example 5: Effect of Secretome on Wound Closure in an Ex Vivo Model of Healing

Pig ear skin explants are maintained in a survival environment. Six-millimeter biopsies are performed at the center, in which a 3-mm annular wound is made. This wound damages the entire epidermis and upper dermis. The products to be evaluated are applied topically. After 48 hours, the biopsies are frozen and the sections are stained with hematoxylin-eosin. The kinetics of re-epithelialization is assessed according to the following method:

1) Assignment of a score defined from 0 to 3 according to the protocol developed by J. Brandner (Pollok et al., 2010, J Cell Mol Med).

Precise measurement in µm of the degree of re-epithelialization from the wound margin. Student's t-test is used to determine significance.

2) The morphological appearance at the edge of the wound is also assessed in order to assess the quality of tissue preservation.

Preparations of products to be applied: the secret was added at a content of 0.4; 0.6 and 1% wt. into the same base preparation as the control repair cream preparation (Cicalfate® cream) analyzed in parallel. Re-epithelialization is evaluated in comparison with this control cream.

The results show the effect of the secretome dose on the progression of wound re-epithelialization. The preparation containing 1% secretome significantly accelerated the progression of wound closure compared to the control repair cream (p = 0.043). In addition, tissue morphological analysis shows excellent tolerance of preparations containing secretions. The results are shown in FIG. 4.

Typical composition: oil-in-water emulsion

secret according to example 2: 0.1-5% glycerol: 1-30% hexylene glycol: 0.1-7% cetearyl glucoside: 0.1-1% cetearyl alcohol: 0.9-4% stearic acid: 0.5-4% glyceryl stearate: 0.1-4% vegetable oil: 0.1-10% water qs (how much it takes to) 100%

SEQUENCE LIST

<110> PIERRE FABRE DERMO-COSMETIC

<120> Bacterial secretion for use in the treatment of skin lesions

<130> B373870PCT D37050

<140> PCT/EP2018/058929

<141> 2018 - 04 - 06

<150> FR1753012

<151> 2017 - 04 - 06

<160> 2

<170>PatentIn version 3.5

<210> 1

<211> 1487

<212> DNA

<213> artificial

<220>

<223> Novel bacterium LMB64, class Beta-proteobacteria, originating from groundwater

<400> 1

agtttgatca tggctcagat tgaacgcggg cggcatgctt tacacatgca agtcgaacgg 60

cagcacgggc ttcggcctgg tggcgagtgg cgaacgggtg agtaatgcgt cggaacgcgc

cgagtagtgg gggataacgc agcgaaagct gtgctaatac cgcatacgta ctgaggtaga 180

aagtggggga ccttcggggcc tcacgctatt cgagcggccg acgtctgatt agctagttgg 240

tggggtaaag gcccaccaag gcgacgatca gtagcgggtc tgagaggatg atccgccaca 300

ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat tttggacaat 360

gggcgcaagc ctgatccagc catgccgcgt gtctgaagaa ggccttcggg ttgtaaagga 420

cttttgtccg ggagcaaagc ctgcttgtta ataccgagtg gggatgagag taccggaaga 480

ataagcaccg gctaactacg tgccagcagc cgcggtaata cgtagggtgc aagcgttaat 540

cggaattact gggcgtaaag cgtgcgcagg cggttgtgca agtctgatgt gaaagccccg 600

ggctcaacct gggaacggca ttggagactg cacggctaga gtgcgtcaga ggggggtaga 660

attccacgtg tagcagtgaa atgcgtagag atgtggagga ataccgatgg cgaaggcagc 720

cccctgggat gacactgacg ctcatgcacg aaagcgtggg gagcaaacag gattagatac 780

cctggtagtc cacgccctaa acgatgtcaa ttagctgttg ggggtttgaa tccttggtag 840

cgaagctaac gcgtgaaatt gaccgcctgg ggagtacggc cgcaaggtta aaactcaaag 900

gaattgacgg ggacccgcac aagcggtgga tgatgtggat taattcgatg caacgcgaaa 960

aaccttacct gctcttgaca tgtaccgaag cctgaagaga tttgggtgtg cccgaaaggg 1020

agcggtaaca caggtgctgc atggctgtcg tcagctcgtg tcgtgagatg ttgggttaag 1080

tcccgcaacg agcgcaaccc ttgtcattag ttgccatcat ttggttgggc actctaatga 1140

gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aagtcctcat ggcccttatg 1200

agcagggctt cacacgtcat acaatggtcg gtacagaggg tcgccaagcc gcgaggtgga 1260

gccaatctca gaaagccgat cgtagtccgg atcgcactct gcaactcgag tgcgtgaagt 1320

cggaatcgct agtaatcgca gatcagcatg ctgcggtgaa tacgttcccg ggtcttgtac 1380

acaccgcccg tcacaccatg ggagtggggg ataccagaag tgggtaggct aaccgcaagg 1440

gaggccgctt accacggtat gcttcatgac tggggtgaag tcgtaac 1487

<210> 2

<211> 10948

<212> DNA

<213> artificial

<220>

<223> Novel bacterium LMB64, class Beta-proteobacteria, originating from groundwater

<400> 2

gggcgcagca ccatcgccca ggccaaaagc cacccgtgcc gattcggcgg cctgttgctc 60

gcgctcaatg cgctgcgcct ggtcggccaa gtcggcggct tgctgctcgg ccttgcccgc 120

cagggtgtca cgctcgcggg tcagttccgc cacctgctcg gctagggcat cgcgctcggc 180

ctcgatcacc tcaccagcgg ccgccagctc ggcggcttcg ctctgcgcct ggaccaagcg 240

gccctcgacc tcggcgcggg cctgggcggc tgcgcgctcg atctcggcgg caatggcggc 300

ggtcaatgcc tggggcagtt ccggcgcagc agcagcggcc accggccgggg cctctcgcca 360

ggcagttaga tgcttgtgaa cggtattcgg gctgccggtg cccaaacgct cgcggatcgc 420

gcggatagtc ggctgctgcc cttcgccgac cagcgcatca gcggcggcgg cgaacgggtg 480

gcccgtgcag aggcccgcgt cgagcagatc gagcagcagg ccgccaaggc ggcacaggcc 540

cacgaagaag cccgcgccgc cgccacgcag gaagcccggc aagtgcaagc cgaacgcgac 600

gaagcccgca aggtggccgc cgaggcgcgc gagcagaccg cgcgcctggc tgggcaactc 660

gaagccctca ccgcgaagga gcgaaaagcc gatgaaagac cgtgaccaca acgaagcgat 720

ggccggcatg tttcaggccg acccccgatt tgccgccgac tatctgcgcc aggtgttggc 780

840

cagtcaggcc gccgcgccga ccgattctgc gccttcggcg ggcctctttg accgggccgg 900

cgtgcgctac gaagtggcgt gcgatgtgat cggggcgttg attgcccatt acgccgaaat 960

catgggacgg gaacgcgagc aggcgcagcc gaatgaggcg gttttgcgcg tggccggagc 1020

catgaaggcg gcgctggccg gggagcggga tgatctcgat ccgcgcgata gcgccggcat 1080

cgaggcggcg atttcgcgct atgcgccact ggcgcgccgg ctgtatggcc aggctgaaaa 1140

cgaccacgcc cgccaggaac agcgtcgcgc cgatttcgac caggtgcatg cttcgctggc 1200

cttggagggg ctggccatga gcgcggacga tttggcggtt caggcgctgc tgatccgggg 1260

cgacattacc cacgatgagg cggtgcagtg ctaccgcatc ctgcatcgcc atgcgcggta 1320

aaaccgactg gccgcccgag gtggccggtg tgctgggttt gctcgaatcg cagccgccag 1380

aggcggcgat gctggtgggc tgttttctgg cggcggtagc ccatcccgat catgcggccg 1440

aattggcgat gttcgacaaa ttgccgccag cggcccggat ggtcgtcggg cgatttttcc 1500

tcgtttttct ggcgggcggc ctggacgatg ccgggcgcga aaaactgcat tcgcacatgc 1560

aggcatggtt tgtccgtcag cgccgttttc ggtgaatggc ttgcctcatc cactagggcc 1620

gggcaagggg tgaacagcgg gcgatgctgg cttgcgggac gaccccgcac ccccggaaaa 1680

cttgtcacac accacgcaac tcccgttgct tcgctaaaag ccttgtgccg caaggctttt 1740

agcgaggcga caccgaagac acatcgcggc gacaccgaaa gggccgaacc ggcctaaaac 1800

ccttgctgcg caaggagaac agccgcgctt tcgcgcgcga aagtgcttca aatgcctgtc 1860

ggcatcagca gggtatggat cggcacgccg aaggcgttgg cgatgcgctc caggttgtcc 1920

aaggagatgt ttctgacttg gcgctcgcag tgcgcaacga aggtgcgatg taggccgcac 1980

tcaaaggcga gcgcttcttg tgaccagcct ctttcccggc gtaatcggat catgttggcc 2040

gcgagcacgg cccgcgcgga atgcgcggtt ggtgcgggag gttgagcggc gggagacatg 2100

2160

tctcatagag aaagacggcg tgacgatggg cagaaaaaca gcaatcaggc gagggggtgc 2220

cgtgttggcc agcctgttga tttgcgcaat tgaaccggtc ggcgcggcct ccctggtcgg 2280

cggtcaaacc gatgattccg tgtgcgacct gggcagcgcg ccacagaacg ccgggaagct 2340

gtcggcagcg ggcgacttca tccgcgcgca gtgcaaaaac ggtcaaatgt tggtgggttc 2400

cggcatcgtg cctgccggcg ggtttgactc ggaagtggtg cgcctggcgc gcaccttctg 2460

ccgcatggcg gacattcaga ctcggcgcac gcagggcaac atggccggcc tcgtcatgga 2520

gatcgacgag gtacggtgca tcatcgggaa gttgccgaca tgagaaaagc gatgttggct 2580

ggctttctgg ccgttgtggt ggccaacgtg gttgccgctg agggcggtgc gcccttgcgc 2640

ggcggtgttt gcatcggacc gttccgtggc gctgattccg tcgtgcactg cgagcacatc 2700

ggcaaggtga cgatccgcca gatttacgaa aagggctttc gggtcgttca catgcaggac 2760

gacaagaaca cagccagcta cgttgtgctg gttatcgagg agcaggcgcg atgagggcga 2820

aagcgtggcg gatgctgttt gccgggcggc gctgggtgct ctggcttccc gtgccggcgt 2880

cggtatggct ggcactgccc gaatggcagc acattcccgc catgttgttg ggcggcctga 2940

tcgtgtggat tcccttctgg ctcgcgtggt ggctcagtga tggcttcgcg ggcatgtcca 3000

ggtggccagg aaccggcgca cctgcggtat ctggcggggt gaacccgcac accggcaagc 3060

catgcacggt gtatcaccag ccgtggggag ataccttcgt gggtggagac tgattatttg 3120

attgaggaac gatgacaagg gccagcaaca agctggccct tgtcgttttc tggactgttt 3180

tacccacaac atccgctctg ctgctgaatt ggcggacatg gcaccgagcc gaacgaacag 3240

aacacgcagc aatccccgg cttggggcgc agcagcgtat ggcaggccgg gcactcgtag 3300

tagaactggc aggcgtccgt gggcatggtt tcctgctgtg cgtggccgca gtgcggggcag 3360

gtcagcacgg attcgagaat gacggcgctc atcgtggcgt tacctctgca cggtggacgg 3420

atagcctgcg ttcgtcgttg ccgaggtcaa cgcttccggc ttggccttgt cggcgtcata 3480

ggtgacggtg gccgttttct tgtcgaaatc gaccttgacg gcgctcacac cgggcacttt 3540

ctccagcgac ttcttgactg tgatcgggca tagctcgcag gtcatgttct gaacggccag 3600

cgtgacggtt ttcggggtgg cggccagcac ggcgagcggc acggcagcca gcagagcaat 3660

cagcagtttg cgcatgggag tctcctttca atagaacagc ggggcgaacc acggcacggc 3720

caacagggca agcaacagca cagtgacgat ccagaacgtc aggcgctgcc gctggcgtgt 3780

gcgcggatca gcgcatggcg tgccgggcgt gcagacctgc ggcaccagat agagcttgcg 3840

gaaggccagt ccgagaaaga gcagcgtcat gccgatgaaa aagggccggt acggctccat 3900

cgcggtcagg ctgccaaccc atgagccacc aatgccaagc accagcagga caagcggccc 3960

gacacagcac accgacgcgc cgatggcggt cagtacgctc acgatcaacg agcttttttc 4020

agtgagtcgt gccatgtcgc tttccttgta cctgtttgcc caagtgttac tctaaatccc 4080

gtacctaagt acggagtcaa gggggtgtga tgggaacaga actgaccatc ggcaagctgg 4140

ctgacgctgc cggggtgaat atcgagacga ttcgctacta ccagcggcgc ggcctgctgg 4200

atgagccgcc taaaccgcca ggggggcatc ggcgctatgc gcctgagcag gcaaaacgtg 4260

tgcgatttat caagcgggca caggcgcttg gtttcacgct ggacgaggta ggcgcgctac 4320

tgaccctgga tgcggcctgc gcctgcggtg agacgcgagc gctggctgtg cgcaagctgg 4380

gtctgatcga gcagaagatg gctgacctcg cggccatgcg gcaggcgctg ggtggattgg 4440

tgcagcagtg tgatgcgggc gacggtggag ccagctgtcc catcatcgac gtgctggcag 4500

gtaattagat gtgttcaaaa aatggtggtt ttctggacac atgccggttt gccctgtcct 4560

gagttgtcct gatgcgttaa agtgttcatt tattcgttca gctttcaatg tggcggaact 4620

gttcatgaat caacgcatcg gctatgcccg cgtttcgacc gacgaccaaa acctagacct 4680

gcaacgggac gcactccggc aggctggatg ctcaaccatt tacgaggaag cagccagcgg 4740

aaagagcgca gcaaggcccg agcttgagca gtgtcggaag gctctccggc ccggcgacac 4800

gcttgtggtg tggcggcttg atcgccttgg gcgcagcctg cccgacctgg tgcagatcgt 4860

ggctgatctt gaacagcgcg gcgtgcattt cgagagcctg accgagaaga tcgagacggg 4920

gagcgcagcg ggtaagctgc aattccatgt tttcgctgca ctcgccgagt tcgagcgcgg 4980

cctgatccgg gagcgaaccc gggcagggct ggatgcagct cgcgcccgtg gccgatccgg 5040

tggacgcaaa ccgaagctgg acgccaagca gatacgccac attaaggcgc tactacgtga 5100

cccgaatacc tgtgttgctg aactcgcccg tgactacggc gtgtcgagaa caactatcta 5160

taaacactgc ggtgtggttc tgccgcgtac agccgatgaa ggggcaatat gacaaaaaag 5220

acaacagcat tcgatgtatt cgagaaatgc gtccaagcag ttcaggctgg tgaactgatc 5280

gaatccgttt ctgcgaagga caaggaattc catttccaga actggtttca gaagcgcctc 5340

cagagcctgt cgatgcactt cgaggggtcg ggtcgcaaca cctacccgga cttctgcttg 5400

gtagagcaca ccgagggcta cgagatcaag ggtttggcat ggcctggccg cgagcgcgac 5460

tacgactcga acagccaagt gccgactggc tatcacaacg gccgtcaaat cttctacgtg 5520

ttcgggcgct accccgcaga cctgtctggc tatgccgatc agggcaacgg ccgcaggcag 5580

tacccggtgg ttgacctcgt ggtctgccac ggcgacttcc tcaacgccga tcacaactac 5640

gtccataaga acaagagcgt aaagggcttt ggcacctacg gcgacatcat gatccgcgac 5700

cggaagatgt acgtcgcgcc gacgccattt gcgctgaccg aaggcaccac tggcctgatg 5760

actttgatcc tgccggagaa cttcggcacc gatgaccgtt accaggtggt cggtaacctc 5820

actcgcgtcg aggcggaaac gctggtggtt ggctacaact ttgacctgcg cacgaacgag 5880

ctgagcgcag agcgcgtgcc caatcccaac gcaggcaccc agcaccgatt cgtggcctac 5940

cggctcaagg atcaagcgag caagcctgtc tccatgactg gcacccaggt gcagcccgac 6000

gagaacaacc tgccggacga cgaatgaaca ccatcaccga caagatcggg ttcgcttacc 6060

cggttgcagc gaccgcgctg gagtgcgact tcccgctggt cgaaatcagc cagatcgccg 6120

agcaggaaag ttggcgaaag gagatcaaca ggccgatcta ccacatccac aagtggtggg 6180

cgaccagact tgggtcggtg tttcgtggca ttacccttgg tgctttgagt cagcctggta 6240

ctgacctctg ggcgcagttc tacaaaacgc acgacctggc cggtaaggta gtgctcgatc 6300

ccttcatggg cagtggcacg acgcttggcg aggccgtcaa gctgggtgcc aaggccatcg 6360

gctgcgacat caacccagtc agtaccttcc tcgtacgtca ggcgttcacg ccggcgtccg 6420

aggcagagct gcgtgccgct ttcgagcggc tggaacgtga cgtggcaccg gagattcggc 6480

gctactacca gacgcgcgat cctaagacgg gcgagctgat tcaggtcttg tactacttct 6540

gggtcaagac ggtgacgacg cccgagggcg aggtaatccc cttgctgtcg cgctacgtgt 6600

tttcacaaga cgcctacccg aagaagaagc cgcgagcgca gatcgtgtgc cctggctgct 6660

ggagtgtgct ggaggatcgc tacgatgcga ctgacctgca ctgccagcac tgcggccacc 6720

agttcaatcc gcaggaaggc ccggccgctg gtcagtacgt caaaaccaag ggcggtcacc 6780

gttaccgcat caaggaacta ctgccaaagg acggtacgcc gccctctcat cgaatgtacg 6840

cgatgatggc cttgcgagcg gatggatcga aggtctatct gccggtgcgg aatgaggact 6900

tggccctcta cgaggaagcc caagaacgcc ttgctacaga ggcactgccg ctgccgaaaa 6960

cctctgttcg acctggccac aacaccgacc aggcgcgcgg ctacaactac acccaatggc 7020

gcgacttctt caatgcgcgc caactgctgt gccttggcct gctgctgcgg gaaatcctga 7080

ccatcgacga cctggcagtg caagagcaga tgctgtgctt gttctccagc accttggagt 7140

tcaacaacct gttttgcagc ttcaagggtg agggaacagg ggccgtgcgg catatgttct 7200

cgaaccacat cctcaagcca gagcgcaccc cgctggagaa ctccgtgtgg ggcactggca 7260

agagcagcgg tacgtttagc acgttgttcg agtctcgcct gctacgtgcg aagcgctacc 7320

tcgatgagcc gttcgagatc gcgttcgagc atgaccagga cggtaaccgc gcaggctcgc 7380

gcaagacggt ggctagccat ccgatccgcg cccgtcgcgt cgaaacctgg ccggaattgg 7440

aggccgcaga tcatggcctg ctgatcctca acggtgacag ctcgaagctg ccggtgcccg 7500

ctggttcggt ggatgccgtg gtgactgatc cgccctactt cgacttcgtt cattactcgg 7560

agttgagcga cttctttttt gcttggctca cccctgtgct gcgccagcgc tatccgtgga 7620

tggcccgcga ggactcgtct gaccaagggg aggtgcagca caaagaccct cgtgtgttcg 7680

7740

tggcgttcag cttccaccac tcgcgtgccg agggctgggc ggccatctat gaagcgatca 7800

acaaggcggg cctggccgta gttgcggctc accctgtcca tgccgagctg cgcgcggcaa 7860

gtcccaagac tgcggccaaa gacccgatca gccttgatgc gattctggtg tgtcgcaaaa 7920

aggcgtttgc cctgcaccag tcgcctgcta tccaggatgt ccgccaggct gttgatgcgc 7980

tggcatcacg gctgcaagct gctggccttc gcatctcggc gggtgaccgc ttcgtgatcg 8040

gcgcagcgca aaccttgatt gcacgcgctg ctgatgacat gggcttcgac gagatcaagg 8100

ttgatcttga ggcaattcgg ctggccgtgg ggccaagggc tgcaacatca aaggctgcga 8160

gtgcgtggga tgacgatgtg cccttctgat tggctgcacg gccttgtcgg cgcatgcgtt 8220

ttgatggcag ccgctgcacg caagccgcgt ccctccgcgt aaagttcatt tatacgcaaa 8280

tacgtatttg cgtgatacaa taacgccata ttaatggagg tgcgtaaatg cggactattg 8340

ttgtggctag ccaaaaaggt ggcgtcggca agacaacgat tgcaggtcac ttgggtgtca 8400

tggccgagca gagcaaagag gggccagtgg cgctgatcga cacagaccca caaggctcgc 8460

tcgcgtcctg gtggaatgag cgaaccaatg aggcaccgct gtttgcacgg gtggaaatcg 8520

gcaagctgac cgagcacctt caggcattgt ccaagggtgg catcaagctg gccatcatcg 8580

acaccccgcc ctctgttacg gaaatgattc agcaggtgct ccgcaccgcc gacttggtac 8640

tgatccccac caggccgagt ccgcatgact tgcgcgcggt cggatctacc gtcgaactgg 8700

tggagaacgc aggcaagcga atgatcttcg tcatcaatgg ggcggcacct cgcgcgcgga 8760

tcgcgggtga ggctgccgtg gcgctttcgc agcatggcac ggttgccccc gtgacgctgt 8820

accagcgcac cgacttcgcc agctcgatga tcgacggccg caccgtccag gaaatcgacc 8880

ccaaggggcg gtcggccgaa gaaatcgggc agttgtggaa atacgtatct acacaactgc 8940

gtaaaatttg atataatacg tacatgcgta ttaatggaga tacgtaaatg gctaaaactg 9000

catctttgac tgccggcctg gtggccaaga aaggggaggc gtcccctgct acggttgtcg 9060

cggcacccca ggttcaacct atcgaagtga aggcatcggc gactggcggc ggccgggatt 9120

actacaaggc gttgaccgtc aagttggatc gtgaccgcta cgagagtctg aaaagcatgg 9180

gcgtgaagct ggacaagaag agcggaaa tctttgtcga ggccctggat ttgtggatga 9240

agtcggccgc tggccagcaa cacgcctaag aggcccctat gcgttcagtg cgctctgccg 9300

tcgaactcgc caaggagttg gccgaaaaag ccaaggcccg ccgcctagcg gcggaaaaga 9360

acgagctggg acttgaaggc ccggcgcagg gcaacgccgg caccactccc agcccggtga 9420

aggttgcggc cgaagtggtg ggcgagcagc cggcacgacg caagggagcg ccgaaagggc 9480

cgcgtggcct gatgccggtg catcatccaa accgcgattt cttcttgtgc gatctgtttg 9540

actacgccct aaaggatgac ggcgtgagca tggaggcccc catcttcacc ttggcaacca 9600

ggccggacac ctctgtttgg cattgggaaa gcaaggatgg gacacgcgcc atcaccgtca 9660

9720

gccagatgac cgaggctatc aatcgcggtc ggcctgatgc gaacaatcga accgtgcgct 9780

ttcgcgtcta tgactacttg gtctcaacca acaagccgac tggcggcaag gagtaccagc 9840

gactggagga tgccctagac aggctgcggg gtacatcgat caagacgaac atcaagacgg 9900

gtggccagcg tgtgaaggaa ggcttcggca tcgtcgatag ctggacgatc atcgagaagg 9960

cccccgacga tgaccgcatg attgccgtcg aggtcacgct ctccaagtgg cttttcaatg 10020

10080

ttgagcgccg tttgtacgag ctggccagga agcactgtgg cgaccaggcc ttttttgtga 10140

ttgggctgga actgttgcag gacaagtgcg gcagcaagtc ggcactgttc gagttccgcc 10200

gtgccttgcg cgagatcatc aaggccgaca ccttgccaga ctaccgcatg acgcttgatg 10260

acgagaaaga ccaggtgatt ttctacaccc gcgacacgaa gaagctagcg gcgtctaccg 10320

ctctggcccg gcgcttccag tgacgcccaa agtattgacg gtcaatactt cgttatttca 10380

cccatgcggt gttaccgctg cgtgttggac gttcccttga cctagcggcc gaggcaggggc 10440

tttcgcgctt tgcattgagc caccaagtgc gtctcgctcc ttcgagcatc aagccctaac 10500

gcgtttcatg tcactttcgc gcacgaaagt cgaggcaaga ggcttgatcg tgtctatcgt 10560

tacatcaccc atgcctgtgg atggacacgt tacatcaccc atgttttctg tggatgggca 10620

cgttacatca cccatacctc acttcgttac atcgcccatg cagcgatttg tggaagcctt 10680

gagcagcaag gctttacgag cgttatccac agccgtaaca cgcgcgcgcg attttttaac 10740

tttataaatc tttaacgcgg ttgcggacaa agcccgcgcc gcctcttggg ggctacgccc 10800

10860

tccccgaggg gtttcgcttc gctgcacccc tcgcgcttcg cgctcacccg catatcgagg 10920

cccccaaagg gggccggatg gtgccccc 10948

Claims (15)

1. The use of bacterial secretome of a bacterium deposited with the CNCM (National Collection of Microorganism Cultures) April 8, 2010 under the number I-4290, in the treatment of skin lesions, moreover, the specified secret is obtained by a method comprising the following stages: a) cultivating said bacterium in a culture medium and under suitable conditions for its growth, b) liquid/solid separation and separation of the liquid phase, where said liquid phase contains a secret, c) adding a basic buffer selected from Tris buffer, arginine buffer and mixtures thereof, to the liquid phase obtained in step b), and incubating the obtained buffered liquid phase for 1-7 hours at a temperature of 1 to 6°C, and obtaining said secretome containing biomolecules secreted by said bacterium into the liquid phase. 2. Use according to claim 1, wherein step b) of liquid/solid separation is followed by step b') of clarifying the liquid phase or the buffered liquid phase by filtration. 3. Application according to claim. 1, where the filtering is carried out with a cut-off threshold of 0.2 μm. 4. Application according to one of paragraphs. 1-3, where said biomolecules consist of peptides, proteins and secondary metabolites secreted by said bacterium. 5. Application according to one of paragraphs. 1-4, where the bacterium contains at least one plasmid containing the sequence of SEQ ID NO: 2 or any sequence having at least 80% identity with the sequence of SEQ ID NO: 2. 6. Application according to one of paragraphs. 1-5, where skin lesions are selected from the group consisting of abrasions, burns, sunburn, scratches, cuts, scrapings, stitches. 7. Application according to one of paragraphs. 1-6 where the skin lesions occur after trauma, surgery, a dermatological procedure, or a cosmetic medical procedure. 8. Application according to one of paragraphs. 1-7, where the treatment of skin lesions is to improve the healing and repair of the skin. 9. Dermatological composition for use in the treatment of skin lesions, containing an effective amount of a bacterial secretome, as defined in one of paragraphs. 1-5 and a dermatological excipient suitable for topical application. 10. Dermatological composition according to claim 9, additionally containing a copper salt and/or a zinc salt.

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