FR3049462A1 - USE OF EXTRACTS OF DEINOCOCCUS BACTERIA AS COSMETIC OR PHARMACEUTICAL AGENTS - Google Patents

Abstract

The present invention relates to a cell extract of bacterial cells of the genus Deinococcus, as well as its use for the preparation of a cosmetic and / or dermatological composition

Description

The present invention relates to the field of cosmetology or dermatology and more particularly to new agents having remarkable properties for cosmetic or dermatological formulations. Already present on Earth 3.5 billion years ago, Deinococci are spherical-like bacteria that measure less than 5 microns in diameter. In the mature state, they are in the form of four red-pink contiguous cells because of their richness in a carotenoid pigment, deinoxanthin.

To survive the early ages of our planet, the Deinococci have developed incredible resistance to radiation, oxidation, heat, desiccation and cold. These bacteria are able to withstand radiation levels of up to five million rads, 5,000 times the lethal dose of 1,000 rads (10 Gy) that can kill a human.

This resilience is due not only to a particular cell structure but also to a very sophisticated DNA repair system that allows the bacterium to repair double-strand breaks in the genome and gives it high genomic stability.

By their total innocuity vis-à-vis the man or the animal, these bacteria find their applications in a multitude of areas, in particular in the production of compounds of interest, in particular from low cost substrate such as plant biomasses (international patent application WO 2009/063079), or as food supplements (international patent application WO 2013/092645).

On the basis of their expertise concerning these remarkable bacteria, the inventors have observed that the cell extracts obtained from Deinococcus bacteria have characteristics that are particularly advantageous for the cosmetic or pharmaceutical industry, in particular to protect the skin against external aggressions such as those caused by by pollution, UV and / or ionizing radiation.

Thus, the subject of the present invention is a cellular extract of bacterial cells of the Deinococcus genus, a process for preparing such an extract, the use of this extract in the preparation of a cosmetic and / or pharmaceutical composition, more particularly a dermatological composition. , and a cosmetic or pharmaceutical composition comprising a cell extract of bacterial cells of the Deinococcus genus, and optionally one or more active agents of cosmetic and / or pharmaceutical interest.

According to a first aspect, the present invention relates to a cell extract of bacterial cells of the genus Deinococcus, also called Deinococci, and a process for preparing this extract.

Bacterial cells of the genus Deinococcus used to produce the cell extract may belong to one or more Deinococcus species. Preferably, the cells belong to a single species of Deinococcus.

The cells may in particular belong to a strain of Deinococcus selected from the group consisting of D. geothermalis, D. cellulolysiticus, D. radiodurans, D. radioresistens, D. proteolyticus, D. radiopugnans, D. radiophilus, D. grandis, D. indicus, D. frigens, D. saxicola, D. maricopensis, D. marmoris, D. deserti, D. murrayi, D. aerius, D. erythromyxa, D. aerolatus, D. antarcticus, D. enclensis, D. guangxiensis, D. puniceus, D. aerophilus, D. aetherius, D. alpinitundrae, D. altitudinis, D. apachensis, D. aquaticus, D. aquatilis, D. aquiradiocola, D. aquivivus, D. caeni, D. claudionis, D. D. depolymerans, D. ficus, D. gobiensis, D. hohokamensis, D. hopiensis, D. misasensis, D. navajonensis, D. papagonensis, D. peraridilitoris, D. pimensis, D. piscis, D. radiomollis, D. reticulitermitis, D. roseus, D. sonorensis, D. wulumuqiensis, D. xibeiensis, D. xinjiangensis, D. yavapaiensis, D. citri, D. guilhemensis, D. phoenicis, D. soli, D. humi, D. saharan , D. mumbaiensis, D. yunweiensis, and any mixture thereof.

The cells may belong to a mesophilic or thermophilic Deinococcus strain, preferably thermophilic.

According to a preferred embodiment, the cell extract is obtained from D. geothermalis cells. Preferably, the cells used are only D. geothermalis cells. Alternatively, the cells comprise a mixture of D. geothermalis cells and cells of one or more other Deinococcus species.

The bacteria used to produce the cell extract may be genetically modified organisms (GMOs) or non-GMOs. Preferably, the bacteria are non-GMOs.

As used herein, the term "cell extract" encompasses the biomasses of Deinococcus bacteria in which the cells are intact, as well as the products obtained from these biomasses by disruption of cell membranes (lysis), fractionation and / or or purification. These products can be obtained by any method known to those skilled in the art, such as, for example, by centrifugation, ultrafiltration, homogenization. microfluidization, milling, lyophilization and / or atomization or any other method described below.

Thus, the present invention relates to a process for preparing a cell extract of bacterial cells of the Deinococcus genus, comprising the cultivation of Deinococcus bacterial cells, the recovery of the biomass thus obtained, and optionally the bursting of the bacterial cells (lysis). .

The Deinococcus bacteria used in the present invention can be cultured according to any method known to those skilled in the art. They may in particular be cultured in a defined synthetic medium or a complex medium, preferably a rich medium that promotes rapid growth. The incubation temperature during the culture depends on the nature of the Deinococcus bacteria used. When these are thermophilic, the cultivation is preferably carried out between 45 ° C. and 48 ° C. When they are mesophilic, the cultivation is preferably carried out between 30 and 37 ° C.

The biomass can be recovered during the exponential phase of growth or during the stationary phase, by any method known to those skilled in the art, in particular by filtration, centrifugation and / or ultrafiltration and / or decantation.

According to one embodiment, the cell extract is a total extract obtained by recovering the biomass after culturing Deinococcus bacterial cells. The biomass can be subjected to additional steps of washing, sterilization, inactivation, dehydration, drying, lyophilization, freezing, bursting of the cells, but does not undergo any fractionation or purification step. In particular, the cell extract may be a bacterial cell lysate of Deinococcus. As used herein, the term "lysate" refers to the product obtained by bursting cells, regardless of the technique employed, particularly by mechanical or non-mechanical methods as detailed below.

According to another embodiment, the cell extract is a partial extract. This extract is preferably a cell lysate supernatant obtained by removal of cell debris after bursting of the cells. The removal of cellular debris can be carried out by any method known to those skilled in the art, for example by centrifugation, filtration, ultrafiltration and / or decantation. Before bursting, the cells may be subjected to additional treatment. They may especially be washed, dehydrated, lyophilized, frozen, inactivated and / or sterilized, for example by autoclaving. The cell extract can also undergo additional fractionation or purification steps, for example to eliminate or purify nucleic acids, eliminate or purify proteins and / or eliminate or purify sugars, eliminate or purify lipids and / or eliminate or purify carotenoids. The bursting of the cells can be achieved by any method known to those skilled in the art for inducing the rupture of cell membranes, and more particularly of the plasma membrane. Such methods are for example described in the article by Chisti and Moo Young, Enzyme Microb. Technol. 1986, vol. 8, April, pp. 194-204. The bursting of the cells may especially be obtained by mechanical methods such as high-pressure homogenization, microfluidization, micro-grinding, sonication, atomization, cavitation or the French press, and / or by non-mechanical methods such as heat treatment (eg desiccation), osmotic shock, enzymatic treatment and / or chemical treatment (eg using detergents, solvents or antibiotics). The cell extract thus obtained can be analyzed by any method known to those skilled in the art, in particular by spectrometry or chromatography methods.

According to a preferred embodiment, the total or partial cell extract according to the invention comprises at least one carotenoid, preferably deinoxanthine and / or one or more quinones, preferably coenzyme Q8. The cell extract may optionally be frozen, lyophilized and / or dehydrated before use.

According to another aspect, the present invention relates to the use of a cell extract according to the invention for the preparation of a cosmetic and / or pharmaceutical composition. It also relates to a process for preparing a cosmetic and / or pharmaceutical composition comprising mixing a cell extract according to the invention with one or more active compounds of cosmetic and / or pharmaceutical interest, and with one or more excipients. or cosmetically and / or pharmaceutically acceptable carriers. It also relates to a cosmetic or pharmaceutical composition comprising a cell extract according to the invention, one or more active compounds of cosmetic and / or dermatological interest, and one or more cosmetically and / or pharmaceutically acceptable excipients or carriers. It also relates to the use of a cell extract according to the invention as a cosmetic or pharmaceutical agent, in particular a dermatological agent.

The pharmaceutical composition is preferably a dermatological composition.

The embodiments described for the cell extract or the process for preparing this extract according to the invention are also to be envisaged in this aspect.

Preferably, the cosmetic or pharmaceutical composition is intended for topical application to the skin, the scalp, the hair and / or the mucous membranes.

The cell extracts according to the invention can be used in any cosmetic or pharmaceutical galenic form suitable for topical application. In particular, the composition may be in the form of cream, gel, simple or complex emulsion (oil in water, water in oil, oil in water in oil, water in oil in water), powder, foam, spray, lotion, oil, solid stick or pencil. The cell extract according to the invention can be used in the composition as a cosmetic and / or dermatological agent. It may especially be used as a moisturizing, antioxidant, anti-UV active agent and / or as a film-forming agent establishing a barrier protecting the skin against external aggressions, for example due to pollution. Π can also be used as an active agent for regenerative, revitalizing, toning, astringent, anti-irritation, disinfectant, detoxifying, soothing, refreshing, healing, cleansing, antioxidant, anti-seborrheic, anti-sweat, anti-UV, anti-redness and / or anti-aging. The cell extract according to the invention may also be used in the composition as a formulation agent, excipient or cosmetically or pharmaceutically acceptable carrier.

The compositions according to the invention generally comprise between 0.001% and 50%, preferably between 0.1% and 20%, by weight of cell extract solids relative to the total weight of the composition.

In the compositions according to the invention, the cell extract according to the invention can be combined with any excipient or carrier usually used in cosmetic or dermatological formulations. Examples of excipients or carriers include, but are not limited to, stabilizers, fatty substances, organic solvents, preservatives, dyes, opacifiers, bulking agents, surfactants, fragrances, propellants, agents and the like. acidifying or basifying agents, polymers or any other compound conventionally used in this type of composition. The cell extract may be associated with one or more active compounds of cosmetic and / or pharmaceutical interest, and more particularly dermatological interest. Preferably, the cell extract is combined with one or more moisturizing, antioxidant, anti-UV, and / or film-forming agents, and / or regenerative, revitalizing, toning, astringent, anti-irritation, disinfectant, detoxifying, soothing agents. refreshing, healing, cleansing, antioxidant, anti-seborrhoeic, anti-sweat, anti-UV, anti-redness and / or anti-aging.

Examples of active compounds include, but are not limited to, moisturizers, antioxidants, anti-radical agents, emollients, UV filters, plant extracts, algae, essential oils, disinfectants, antibiotic, antifungal or antiparasitic agents, astringent agents or agents stimulating cell renewal.

According to the nature of the active agents contained in the composition, it may be a moisturizing composition, regenerative, revitalizing, toning, astringent, anti-irritation, disinfectant, detoxifying, soothing, refreshing, healing, cleansing, antioxidant, anti-seborrheic, anti-exudative, anti-UV, anti-redness and / or anti-aging.

The composition may also be a make-up product, especially a foundation, a lipstick or a mascara.

According to a particular embodiment, the composition is a dermatological composition. This may be intended for the treatment and / or prevention of any skin disorder such as skin infections, a dermatosis such as pruritus, eczema or atopic dermatitis, trauma to the skin or mucosa such as a cut, scratch, burn, irritation, erythema, blister or blister, injury caused by exposure to chemicals, eg detergents, to certain therapeutic agents such as anti-cancer or anti-cancer agents Acne, radiotherapy, laser or some cosmetic processes such as peeling or dermo-abrasion. The composition may also be intended for the treatment and / or prevention of hyperpigmentation (brown spots), acne, and / or skin aging when the severity of these disorders is pathology. Other features and advantages of the invention will appear better on reading the following examples given for illustrative and non-limiting.

Examples

Preparation of a total cellular extract of D.geothermalis

Culture

Colonies of D. geothermalis are subcultured in 25 ml of CMG2% medium (Peptone 2 g / L, yeast extract 5 g / L, 55 mM Glucose (20 g / L), 40 mM MOPS acid, 20 mM NH4Cl, NaOH 10mM, 10mM KOH, 0.5mM CaCl2.2H2O, 0.276mM Na2SO4.10H2O, 0.528mM MgCl2.6H2 O (ΝΗ4) ό (Μθ7) θ24.4Η2θ 3μM, 0.4μM H3BO3, 30μM CoCl2.6H2O; CUSO4.5H2O 10 μM, MnCl 0.25 μM, ZnSO4.7H20 10 μM, D-Biotin 1 μg / L, Niacin (nicotinic acid) 1 μg / L, vitamin B6 1 μg / L, vitamin B, FeCh 20 μM, Sodium Citrate .2H20 20 μM; 5.7 mM K2HPO4) containing 2% glucose as the only carbon source, and cultured at 45 ° C at 250 rpm overnight. Inoculum of the growth phase is used to inoculate 25 ml of the same fresh medium with an OD 600 nm (OD600) of 0.4. The culture is conducted at 45 ° C, 250 rpm overnight and serves as an inoculum to seed a mineral medium defined below at an OD 600 nm (OD600) of 0.4 for the production of cell extract.

The culture for the production of the cell extract is carried out at 45 ° C., 250 rpm for 48 hours in 25 ml of a defined mineral medium containing (NH 4) 2 SO 4 <100 mM; NaH₂PO0.H₂O <10 mM; KCl <10 mM; Na 2 SO 4 <10 mM; Citric acid <30 mM; MgCl 2 · 6H 2 O <10 mM; CaCl2.2H2O <10 mM; ZnCh <50 mg / L; FeSO4.7H2O <50 mg / L; MnCl 2 · 4H 2 O <50 mg / L; CuSO 4 <50 mg / L; C0CI2.6H2O <50 mg / L; H3BO3 <5 mg / L; MES <200 mM; (ΝΗ4) όΜθ7θ24.4Η2θ <0.5 mM; Glucose <30 g / L (166 mM). Biomass recovery

The culture (25 ml) is centrifuged at 4000 rpm, 20 min at 4 ° C.

Preparation of the total cell extract

The bacterial pellet is washed three times with sterile water and is suspended in a buffer containing 10 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5 μM DTT, 0.2 mM EDTA, 10 mg / ml lysozyme, incubated at 37 ° C. C for 40 min and immediately frozen in liquid nitrogen and then broken into a BeadBreader homogenizer (Biospec, 100pm beads). Alternatively, the bacterial pellet may be suspended in 50 μM phosphate buffer and the cells are broken by sonication (at 4 ° C for 60 min with 40 sec pulses spaced 40 sec) or by passage through a press. French (20,000 Ib / in ").

Optionally, cell debris can be removed after centrifugation at 20000g for 30 min at 4 ° C.

Claims (19)

claims 1. Use of a cellular extract of bacterial cells of the Deinococcus genus for the preparation of a cosmetic and / or dermatological composition. 2. Use according to claim 1, wherein the cell extract is a lysate of bacterial cells of the genus Deinococcus. The use according to claim 1, wherein the cell extract is a bacterial cell lysate supernatant of the genus Deinococcus. The use according to any one of the preceding claims, wherein the bacterial cells belong to a strain of Deinococcus selected from the group consisting of D. geothermalis, D. cellulolysiticus, D. radiodurans, D. radioresistens, D. radiotolerans, D. proteolyticus, D. radiopugnans, D. radiophilus, D. grandis, D. indicus, D. frigens, D. saxicola, D. maricopensis, D. marmoris, D. deserti, D. murrayi, D. aerius, D. erythromyxa , D. aerolatus, D. antarcticus, D. enclensis, D. guangriensis, D. puniceus, D. aerophilus, D. aetherius, D. alpinitundrae, D. altitudinis, D. apachensis, D. aquaticus, D. aquatilis, D. Aquiradiocola, D. aquivivus, D. caeni, D. claudionis, D. daejeonensis, D. depolymerans, D. ficus, D. gobiensis, D. hohokamensis, D. hopiensis, D. misasensis, D. navajonensis, D. papagonensis , D. peraridilitoris, D. pimensis, D. piscis, D. radiomollis, D. reticulitermitis, D. roseus, D. sonorensis, D. wulumuqiensis, D. xibeiensis, D. xinjiangensis, D. yavapaiensis, D. citri, D. guilhemensis, D. phoenicis, D. soli, D. swuensis, D. humi, D. sahariens, D. mumbaiensis and D. yunweiensis, and any mixture thereof, preferably a strain of D. geothermalis. 5. Use according to any one of the preceding claims, wherein the composition is intended for topical application to the skin, hair, scalp and / or mucous membranes. 6. Use according to any preceding claim, wherein the composition is in the form of cream, gel, single or complex emulsion, powder, foam, spray, lotion, oil, stick solid or pencil. 7. Use according to any one of the preceding claims, wherein the cell extract is associated with one or more active compounds of cosmetic and / or dermatological interest. 8. Use according to any one of the preceding claims, wherein the composition is a hydrating composition, regenerative, revitalizing, toning, astringent, anti-irritation, disinfectant, detoxifying, soothing, refreshing, healing, cleansing, antioxidant , anti-seborrhoeic, anti-sweat, anti-UV, anti-redness and / or anti-aging. 9. Use according to any one of the preceding claims wherein the composition is a makeup product, in particular a foundation, a mascara or a lipstick. 10. Use according to any one of the preceding claims, wherein the composition is a dermatological composition, in particular for the treatment and / or prevention of cutaneous disorders such as skin infections, dermatitis such as itching. , eczema or atopic dermatitis, trauma to the skin or mucous membrane such as a cut, scratch, burn, irritation, erythema, blister or blister, injury caused by exposure to products chemicals, for example detergents, certain therapeutic agents such as anti-cancer or anti-acne agents, radiotherapy, lasers or even cosmetic procedures such as peels or dermo-abrasion. 11. Use according to any one of the preceding claims, wherein the cell extract is used as moisturizing agent, antioxidant, anti-UV agent, film-forming agent and / or active agent for regenerative, revitalizing, toning, astringent, anti-irritation, disinfectant, detoxifying, soothing, refreshing, healing, cleansing, antioxidant, anti-seborrhoeic, anti-sweat, anti-UV, anti-redness and / or anti-aging. The use according to any one of the preceding claims, wherein the cell extract is used in combination with one or more other moisturizing, antioxidant, anti-UV, and / or film-forming agents, and / or regenerative agents, revitalizing, toning, astringent, anti-irritation, disinfectant, detoxifying, soothing, refreshing, healing, cleansing, antioxidant, anti-seborrhoeic, anti-sweat, anti-UV, anti-redness and / or anti-aging. 13. Use according to any one of the preceding claims, wherein the composition comprises between 0.001 and 50% solids content of cell extract relative to the total weight of the composition. A process for preparing a cell extract of Deinococcus bacterial cells comprising the steps of culturing Deinococcus bacterial cells, and recovering the biomass thus obtained. The preparation method of claim 14, further comprising a step of bursting the cells and / or a step of removing cell debris. Cell extract obtainable by the method of claim 14 or 15. The cell extract of claim 16, said extract being a lysate or a lysate supernatant of bacterial cells of the genus Deinococcus. 18. A process for preparing a cosmetic and / or dermatological composition comprising the mixture of a cell extract according to claim 16 or 17, with one or more active compounds of cosmetic and / or dermatological interest, and with one or more excipients. or cosmetically and / or pharmaceutically acceptable carriers. 19. Cosmetic or dermatological composition comprising a cell extract according to claim 16 or 17, one or more active compounds of cosmetic and / or dermatological interest, and one or more cosmetically and / or pharmaceutically acceptable excipients or carriers.